Transgenic non-human animals for producing chimeric antibodies

ABSTRACT

The invention relates to transgenic non-human animals capable of producing heterologous antibodies and methods for producing human sequence antibodies which bind to human antigens with substantial affinity.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.11/009,873, filed on Dec. 10, 2004 now U.S. Pat. No. 7,501,552, which isa Continuation of application Ser. No. 09/724,965 filed on Nov. 28,2000, issued as U.S. Pat. No. 7,041,871 on May 9, 2006, which is aContinuation-in-part of application Ser. No. 09/042,353 filed Mar. 13,1998, issued as U.S. Pat. No. 6,255,458 on Jul. 3, 2001, which is aContinuation-in-part of application Ser. No. 08/758,417 filed on Dec. 2,1996, issued as U.S. Pat. No. 6,300,129 on Oct. 9, 2001, which is aContinuation-in-part of application Ser. No. 08/728,463 filed on Oct.10, 1996, issued as U.S. Pat. No. 7,084,260 on Aug. 1, 2006, which is aContinuation-in-part of application Ser. No. 08/544,404 filed on Oct.10, 1995, issued as U.S. Pat. No. 5,770,429 on Jun. 23, 1998, which is aContinuation-in part of application Ser. No. 08/352,322 filed on Dec. 7,1994, issued as U.S. Pat. No. 5,625,126 on Apr. 29, 1997, which is aContinuation-in-part of application Ser. No. 08/209,741 filed on Mar. 9,1994, now abandoned, all of which are hereby incorporated by referencein their entireties herein.

SEQUENCE LISTING

The specification further incorporates by reference the Sequence Listingsubmitted herewith via EFS on May 7, 2009 Pursuant to 37 C.F.R.§1.52(e)(5), the Sequence Listing text file, identified as12239523SeqListing.txt, is 170,325 bytes and was created on May 7, 2009.The Sequence Listing, electronically filed herewith, does not extendbeyond the scope of the specification and thus does not contain newmatter.

TECHNICAL FIELD

The invention relates to transgenic non-human animals capable ofproducing heterologous antibodies, transgenes used to produce suchtransgenic animals, transgenes capable of functionally rearranging aheterologous D gene in V-D-J recombination, immortalized B-cells capableof producing heterologous antibodies, methods and transgenes forproducing heterologous antibodies of multiple isotypes, methods andtransgenes for producing heterologous antibodies wherein a variableregion sequence comprises somatic mutation as compared to germlinerearranged variable region sequences, transgenic nonhuman animals whichproduce antibodies having a human primary sequence and which bind tohuman antigens, hybridomas made from B cells of such transgenic animals,and monoclonal antibodies expressed by such hybridomas.

BACKGROUND OF THE INVENTION

One of the major impediments facing the development of in vivotherapeutic and diagnostic applications for monoclonal antibodies inhumans is the intrinsic immunogenicity of non-human immunoglobulins. Forexample, when immunocompetent human patients are administeredtherapeutic doses of rodent monoclonal antibodies, the patients produceantibodies against the rodent immunoglobulin sequences; these humananti-mouse antibodies (HAMA) neutralize the therapeutic antibodies andcan cause acute toxicity. Hence, it is desirable to produce humanimmunoglobulins that are reactive with specific human antigens that arepromising therapeutic and/or diagnostic targets. However, producinghuman immunoglobulins that bind specifically with human antigens isproblematic.

The present technology for generating monoclonal antibodies involvespre-exposing, or priming, an animal (usually a rat or mouse) withantigen, harvesting B-cells from that animal, and generating a libraryof hybridoma clones. By screening a hybridoma population for antigenbinding specificity (idiotype) and also screening for immunoglobulinclass (isotype), it is possible to select hybridoma clones that secretethe desired antibody.

However, when present methods for generating monoclonal antibodies areapplied for the purpose of generating human antibodies that have bindingspecificities for human antigens, obtaining B-lymphocytes which producehuman immunoglobulins a serious obstacle, since humans will typicallynot make immune responses against self-antigens.

Hence, present methods of generating human monoclonal antibodies thatare specifically reactive with human antigens are clearly insufficient.It is evident that the same limitations on generating monoclonalantibodies to authentic self antigens apply where non-human species areused as the source of B-cells for making the hybridoma.

The construction of transgenic animals harboring a functionalheterologous immunoglobulin transgene are a method by which antibodiesreactive with self antigens may be produced. However, in order to obtainexpression of therapeutically useful antibodies, or hybridoma clonesproducing such antibodies, the transgenic animal must produce transgenicB cells that are capable of maturing through the B lymphocytedevelopment pathway. Such maturation requires the presence of surfaceIgM on the transgenic B cells, however isotypes other than IgM aredesired for therapeutic uses. Thus, there is a need for transgenes andanimals harboring such transgenes that are able to undergo functionalV-D-J rearrangement to generate recombinational diversity and junctionaldiversity. Further, such transgenes and transgenic animals preferablyinclude cis-acting sequences that facilitate isotype switching from afirst isotype that is required for B cell maturation to a subsequentisotype that has superior therapeutic utility.

A number of experiments have reported the use of transfected cell linesto determine the specific DNA sequences required for Ig generearrangement (reviewed by Lewis and Gellert (1989), Cell, 59, 585-588).Such reports have identified putative sequences and concluded that theaccessibility of these sequences to the recombinase enzymes used forrearrangement is modulated by transcription (Yancopoulos and Alt (1985),Cell, 40, 271-281). The sequences for V(D)J joining are reportedly ahighly conserved, near-palindromic heptamer and a less well conservedAT-rich nanomer separated by a spacer of either 12 or 23 bp (Tonegawa(1983), Nature, 302, 575-581; Hesse, et al. (1989), Genes in Dev., 3,1053-1061). Efficient recombination reportedly occurs only between sitescontaining recombination signal sequences with different length spacerregions.

Ig gene rearrangement, though studied in tissue culture cells, has notbeen extensively examined in transgenic mice. Only a handful of reportshave been published describing rearrangement test constructs introducedinto mice [Buchini, et al. (1987), Nature, 326, 409-411 (unrearrangedchicken λ transgene); Goodhart, et al. (1987), Proc. Natl. Acad. Sci.USA, 84, 4229-4233) (unrearranged rabbit κ gene); and Bruggemann, et al.(1989), Proc. Natl. Acad. Sci. USA, 86, 6709-6713 (hybrid mouse-humanheavy chain)). The results of such experiments, however, have beenvariable, in some cases, producing incomplete or minimal rearrangementof the transgene.

Further, a variety of biological functions of antibody molecules areexerted by the Fc portion of molecules, such as the interaction withmast cells or basophils through Fcε, and binding of complement by Fcμ orFcγ, it further is desirable to generate a functional diversity ofantibodies of a given specificity by variation of isotype.

Although transgenic animals have been generated that incorporatetransgenes encoding one or more chains of a heterologous antibody, therehave been no reports of heterologous transgenes that undergo successfulisotype switching. Transgenic animals that cannot switch isotypes arelimited to producing heterologous antibodies of a single isotype, andmore specifically are limited to producing an isotype that is essentialfor B cell maturation, such as IgM and possibly IgD, which may be oflimited therapeutic utility. Thus, there is a need for heterologousimmunoglobulin transgenes and transgenic animals that are capable ofswitching from an isotype needed for B cell development to an isotypethat has a desired characteristic for therapeutic use.

Based on the foregoing, it is clear that a need exists for methods ofefficiently producing heterologous antibodies, e.g. antibodies encodedby genetic sequences of a first species that are produced in a secondspecies. More particularly, there is a need in the art for heterologousimmunoglobulin transgenes and transgenic animals that are capable ofundergoing functional V-D-J gene rearrangement that incorporates all ora portion of a D gene segment which contributes to recombinationaldiversity. Further, there is a need in the art for transgenes andtransgenic animals that can support V-D-J recombination and isotypeswitching so that (1) functional B cell development may occur, and (2)therapeutically useful heterologous antibodies may be produced. There isalso a need for a source of B cells which can be used to make hybridomasthat produce monoclonal antibodies for therapeutic or diagnostic use inthe particular species for which they are designed. A heterologousimmunoglobulin transgene capable of functional V-D-J recombinationand/or capable of isotype switching could fulfill these needs.

In accordance with the foregoing object transgenic nonhuman animals areprovided which are capable of producing a heterologous antibody, such asa human antibody.

Further, it is an object to provide B-cells from such transgenic animalswhich are capable of expressing heterologous antibodies wherein suchB-cells are immortalized to provide a source of a monoclonal antibodyspecific for a particular antigen.

In accordance with this foregoing object, it is a further object of theinvention to provide hybridoma cells that are capable of producing suchheterologous monoclonal antibodies.

Still further, it is an object herein to provide heterologousunrearranged and rearranged immunoglobulin heavy and light chaintransgenes useful for producing the aforementioned non-human transgenicanimals.

Still further, it is an object herein to provide methods to disruptendogenous immunoglobulin loci in the transgenic animals.

Still further, it is an object herein to provide methods to induceheterologous antibody production in the aforementioned transgenicnon-human animal.

A further object of the invention is to provide methods to generate animmunoglobulin variable region gene segment repertoire that is used toconstruct one or more transgenes of the invention.

The references discussed herein are provided solely for their disclosureprior to the filing date of the present application. Nothing herein isto be construed as an admission that the inventors are not entitled toantedate such disclosure by virtue of prior invention.

SUMMARY OF THE INVENTION

Transgenic nonhuman animals are provided which are capable of producinga heterologous antibody, such as a human antibody. Such heterologousantibodies may be of various isotypes, including: IgG1, IgG2, IgG3,IgG4, IgM, IgA1, IgA2, IgA_(sec), IgD, of IgE. In order for suchtransgenic nonhuman animals to make an immune response, it is necessaryfor the transgenic B cells and pre-B cells to produce surface-boundimmunoglobulin, particularly of the IgM (or possibly IgD) isotype, inorder to effectuate B cell development and antigen-stimulatedmaturation. Such expression of an IgM (or IgD) surface-boundimmunoglobulin is only required during the antigen-stimulated maturationphase of B cell development, and mature B cells may produce otherisotypes, although only a single switched isotype may be produced at atime.

Typically, a cell of the B-cell lineage will produce only a singleisotype at a time, although cis or trans alternative RNA splicing, suchas occurs naturally with the μ_(S) (secreted μ) and μ_(M)(membrane-bound μ) forms, and the μ and δ immunoglobulin chains, maylead to the contemporaneous expression of multiple isotypes by a singlecell. Therefore, in order to produce heterologous antibodies of multipleisotypes, specifically the therapeutically useful IgG, IgA, and IgEisotypes, it is necessary that isotype switching occur. Such isotypeswitching may be classical class-switching or may result from one ormore non-classical isotype switching mechanisms.

The invention provides heterologous immunoglobulin transgenes andtransgenic nonhuman animals harboring such transgenes, wherein thetransgenic animal is capable of producing heterologous antibodies ofmultiple isotypes by undergoing isotype switching. Classical isotypeswitching occurs by recombination events which involve at least oneswitch sequence region in the transgene. Non-classical isotype switchingmay occur by, for example, homologous recombination between human Σ_(μ)and human Σ_(μ) sequences (δ-associated deletion). Alternativenon-classical switching mechanisms, such as intertransgene and/orinterchromosomal recombination, among others, may occur and effectuateisotype switching. Such transgenes and transgenic nonhuman animalsproduce a first immunoglobulin isotype that is necessary forantigen-stimulated B cell maturation and can switch to encode andproduce one or more subsequent heterologous isotypes that havetherapeutic and/or diagnostic utility. Transgenic nonhuman animals ofthe invention are thus able to produce, in one embodiment, IgG, IgA,and/or IgE antibodies that are encoded by human immunoglobulin geneticsequences and which also bind specific human antigens with highaffinity.

The invention also encompasses B-cells from such transgenic animals thatare capable of expressing heterologous antibodies of various isotypes,wherein such B-cells are immortalized to provide a source of amonoclonal antibody specific for a particular antigen. Hybridoma cellsthat are derived from such B-cells can serve as one source of suchheterologous monoclonal antibodies.

The invention provides heterologous unrearranged and rearrangedimmunoglobulin heavy and light chain transgenes capable of undergoingisotype switching in vivo in the aforementioned non-human transgenicanimals or in explanted lymphocytes of the B-cell lineage from suchtransgenic animals. Such isotype switching may occur spontaneously or beinduced by treatment of the transgenic animal or explanted B-lineagelymphocytes with agents that promote isotype switching, such asT-cell-derived lymphokines (e.g., IL-4 and IFN_(γ)).

Still further, the invention includes methods to induce heterologousantibody production in the aforementioned transgenic non-human animal,wherein such antibodies may be of various isotypes. These methodsinclude producing an antigen-stimulated immune response in a transgenicnonhuman animal for the generation of heterologous antibodies,particularly heterologous antibodies of a switched isotype (i.e., IgG,IgA, and IgE).

This invention provides methods whereby the transgene contains sequencesthat effectuate isotype switching, so that the heterologousimmunoglobulins produced in the transgenic animal and monoclonalantibody clones derived from the B-cells of said animal may be ofvarious isotypes.

This invention further provides methods that facilitate isotypeswitching of the transgene, so that switching between particularisotypes may occur at much higher or lower frequencies or in differenttemporal orders than typically occurs in germline immunoglobulin loci.Switch regions may be grafted from various C_(H) genes and ligated toother C_(H) genes in a transgene construct; such grafted switchsequences will typically function independently of the associated C_(H)gene so that switching in the transgene construct will typically be afunction of the origin of the associated switch regions. Alternatively,or in combination with switch sequences, δ-associated deletion sequencesmay be linked to various C_(H) genes to effect non-classical switchingby deletion of sequences between two δ-associated deletion sequences.Thus, a transgene may be constructed so that a particular C_(H) gene islinked to a different switch sequence and thereby is switched to morefrequently than occurs when the naturally associated switch region isused.

This invention also provides methods to determine whether isotypeswitching of transgene sequences has occurred in a transgenic animalcontaining an immunoglobulin transgene.

The invention provides immunoglobulin transgene constructs and methodsfor producing immunoglobulin transgene constructs, some of which containa subset of germline immunoglobulin loci sequences (which may includedeletions). The invention includes a specific method for facilitatedcloning and construction of immunoglobulin transgenes, involving avector that employs unique XhoI and SalI restriction sites flanked bytwo unique NotI sites. This method exploits the complementary termini ofXhoI and SalI restrictions sites and is useful for creating largeconstructs by ordered concatemerization of restriction fragments in avector.

The transgenes of the invention include a heavy chain transgenecomprising DNA encoding at least one variable gene segment, onediversity gene segment, one joining gene segment and one constant regiongene segment. The immunoglobulin light chain transgene comprises DNAencoding at least one variable gene segment, one joining gene segmentand one constant region gene segment. The gene segments encoding thelight and heavy chain gene segments are heterologous to the transgenicnon-human animal in that they are derived from, or correspond to, DNAencoding immunoglobulin heavy and light chain gene segments from aspecies not consisting of the transgenic non-human animal. In one aspectof the invention, the transgene is constructed such that the individualgene segments-are unrearranged, i.e., not rearranged so as to encode afunctional immunoglobulin light or heavy chain. Such unrearrangedtransgenes permit recombination of the gene segments (functionalrearrangement) and expression of the resultant rearranged immunoglobulinheavy and/or light chains within the transgenic non-human animal whensaid animal is exposed to antigen.

In one aspect of the invention, heterologous heavy and lightimmunoglobulin transgenes comprise relatively large fragments ofunrearranged heterologous DNA. Such fragments typically comprise asubstantial portion of the C, J (and in the case of heavy chain, D)segments from a heterologous immunoglobulin locus. In addition, suchfragments also comprise a substantial portion of the variable genesegments.

In one embodiment, such transgene constructs comprise regulatorysequences, e.g. promoters, enhancers, class switch regions,recombination signals and the like, corresponding to sequences derivedfrom the heterologous DNA. Alternatively, such regulatory sequences maybe incorporated into the transgene from the same or a related species ofthe non-human animal used in the invention. For example, humanimmunoglobulin gene segments may be combined in a transgene with arodent immunoglobulin enhancer sequence for use in a transgenic mouse.

In a method of the invention, a transgenic non-human animal containinggermline unrearranged light and heavy immunoglobulin transgenes—thatundergo VDJ joining during D-cell differentiation—is contacted with anantigen to induce production of a heterologous antibody in a secondaryrepertoire B-cell.

Also included in the invention are vectors and methods to disrupt theendogenous immunoglobulin loci in the non-human animal to be used in theinvention. Such vectors and methods utilize a transgene, preferablypositive-negative selection vector, which is constructed such that ittargets the functional disruption of a class of gene segments encoding aheavy and/or light immunoglobulin chain endogenous to the non-humananimal used in the invention. Such endogenous gene segments includediversity, joining and constant region gene segments. In this aspect ofthe invention, the positive-negative selection vector is contacted withat least one embryonic stem cell of a non-human animal after which cellsare selected wherein the positive-negative selection vector hasintegrated into the genome of the non-human animal by way of homologousrecombination. After transplantation, the resultant transgenic non-humananimal is substantially incapable of mounting an immunoglobulin-mediatedimmune response as a result of homologous integration of the vector intochromosomal DNA. Such immune deficient non-human animals may thereafterbe used for study of immune deficiencies or used as the recipient ofheterologous immunoglobulin heavy and light chain transgenes.

The invention also provides vectors, methods, and compositions usefulfor suppressing the expression of one or more species of immunoglobulinchain(s), without disrupting an endogenous immunoglobulin locus. Suchmethods are useful for suppressing expression of one or more endogenousimmunoglobulin chains while permitting the expression of one or moretransgene-encoded immunoglobulin chains. Unlike genetic disruption of anendogenous immunoglobulin chain locus, suppression of immunoglobulinchain expression does not require the time-consuming breeding that isneeded to establish transgenic animals homozygous for a disruptedendogenous Ig locus. An additional advantage of suppression as comparedto endogenous Ig gene disruption is that, in certain embodiments, chainsuppression is reversible within an individual animal. For example, Igchain suppression may be accomplished with: (1) transgenes encoding andexpressing antisense RNA that specifically hybridizes to an endogenousIg chain gene sequence, (2) antisense oligonucleotides that specificallyhybridize to an endogenous Ig chain gene sequence, and (3)immunoglobulins that bind specifically to an endogenous Ig chainpolypeptide.

The invention provides transgenic non-human animals comprising: ahomozygous pair of functionally disrupted endogenous heavy chainalleles, a homozygous pair of functionally disrupted endogenous lightchain alleles, at least one copy of a heterologous immunoglobulin heavychain transgene, and at least one copy of a heterologous immunoglobulinheavy chain transgene, wherein said animal makes an antibody responsefollowing immunization with an antigen, such as a human antigen (e.g.,CD4). The invention also provides such a transgenic non-human animalwherein said functionally disrupted endogenous heavy chain allele is aJ_(H) region homologous recombination knockout, said functionallydisrupted endogenous light chain allele is a J_(κ) region homologousrecombination knockout, said heterologous immunoglobulin heavy chaintransgene is the HC1 or HC2 human minigene transgene, said heterologouslight chain transgene is the KC2 or KC1e human κ transgene, and whereinsaid antigen is a human antigen.

The invention also provides various embodiments for suppressing,ablating, and/or functionally disrupting the endogenous nonhumanimmunoglobulin loci.

The invention also provides transgenic mice expressing both humansequence heavy chains and chimeric heavy chains comprising a humansequence heavy chain variable region and a murine sequence heavy chainconstant region. Such chimeric heavy chains are generally produced bytrans-switching between a functionally rearranged human transgene and anendogenous murine heavy chain constant region (e.g., γ1, γ2a, γ2b, γ3).Antibodies comprising such chimeric heavy chains, typically incombination with a transgene-encoded human sequence light chain orendogenous murine light chain, are formed in response to immunizationwith a predetermined antigen. The transgenic mice of these embodimentscan comprise B cells which produce (express) a human sequence heavychain at a first timepoint and trans-switch to produce (express) achimeric heavy chain composed of a human variable region and a murineconstant region (e.g., γ1, γ2a, γ2b, γ3) at a second (subsequent)timepoint; such human sequence and chimeric heavy chains areincorporated into functional antibodies with light chains; suchantibodies are present in the serum of such transgenic mice. Thus, torestate: the transgenic mice of these embodiments can comprise B cellswhich express a human sequence heavy chain and subsequently switch (viatrans-switching or cis-switching) to express a chimeric orisotype-switched heavy chain composed of a human variable region and aalternative constant region (e.g., murine γ1, γ2a, γ2b, γ3; human γ, α,ε); such human sequence and chimeric or isotype-switched heavy chainsare incorporated into functional antibodies with light chains (human ormouse); such antibodies are present in the serum of such transgenicmice.

The invention also provides a method for generating a large transgene,said method comprising:

introducing into a mammalian cell at least three polynucleotide species;a first polynucleotide species having a recombinogenic region ofsequence identity shared with a second polynucleotide species, a secondpolynucleotide species having a recombinogenic region of sequenceidentity shared with a first polynucleotide species and a recombinogenicregion of sequence identity shared with a third polynucleotide species,and a third polynucleotide species having a recombinogenic region ofsequence identity shared with said second polynucleotide species.

Recombinogenic regions are regions of substantial sequence identitysufficient to generate homologous recombination in vivo in a mammaliancell (e.g., ES cell), and preferably also in non-mammalian eukaryoticcells (e.g., Saccharaomyces and other yeast or fungal cells). Typically,recombinogenic regions are at least 50 to 100000 nucleotides long orlonger, preferably 500 nucleotides to 10000 nucleotides long, and areoften 80-100 percent identical, frequently 95-100 percent identical,often isogenic.

The references discussed herein are provided solely for their disclosureprior to the filing date of the present application. Nothing herein isto be construed as an admission that the inventors are not entitled toantedate such disclosure by virtue of prior invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the complementarity determining regions CDR1, CDR2 andCDR3 and framework regions FR1, FR2, FR3 and FR4 in unrearranged genomicDNA and mRNA expressed from a rearranged immunoglobulin heavy chaingene,

FIG. 2 depicts the human λ chain locus,

FIG. 3 depicts the human κ chain locus,

FIG. 4 depicts the human heavy chain locus,

FIG. 5 depicts a transgene construct containing a rearranged IgM geneligated to a 25 kb fragment that contains human γ3 and γ1 constantregions followed by a 700 bp fragment containing the rat chain 3′enhancer sequence.

FIG. 6 is a restriction map of the human κ chain locus depicting thefragments to be used to form a light chain transgene by way of in vivohomologous recombination.

FIG. 7 depicts the construction of pGP1.

FIG. 8 depicts the construction of the polylinker contained in pGP1.

FIG. 9 depicts the fragments used to construct a * human heavy chaintransgene of the invention.

FIG. 10 depicts the construction of pHIG1 and pCON1.

FIG. 11 depicts the human Cγ1 fragments which are inserted into pRE3(rat enhancer 3′) to form pREG2.

FIG. 12 depicts the construction of pHIG3′ and PCON.

FIG. 13 depicts the fragment containing human D region segments used inconstruction of the transgenes of the invention.

FIG. 14 depicts the construction of pHIG2 (D segment containingplasmid).

FIG. 15 depicts the fragments covering the human J_(κ) and human C_(κ)gene segments used in constructing a transgene of the invention.

FIG. 16 depicts the structure of pEμ.

FIG. 17 depicts the construction of pKapH.

FIGS. 18A through 18D depict the construction of a positive-negativeselection vector for functionally disrupting the endogenous heavy chainimmunoglobulin locus of mouse.

FIGS. 19A through 19C depict the construction of a positive-negativeselection vector for functionally disrupting the endogenousimmunoglobulin light chain loci in mouse.

FIGS. 20A through 20E depict the structure of a kappa light chaintargeting vector.

FIGS. 21A through 21F depict the structure of a mouse heavy chaintargeting vector.

FIG. 22 depicts the map of vector pGPe.

FIG. 23 depicts the structure of vector pJM2.

FIG. 24 depicts the structure of vector pCOR1.

FIG. 25 depicts the transgene constructs for pIGM1, pHC1 and pHC2.

FIG. 26 depicts the structure of pγe2.

FIG. 27 depicts the structure of pVGE1.

FIG. 28 depicts the assay results of human Ig expression in a pHC1transgenic mouse.

FIG. 29 depicts the structure of pJCK1.

FIG. 30 depicts the construction of a synthetic heavy chain variableregion.

FIG. 31 is a schematic representation of the heavy chain minilocusconstructs pIGM1, pHC1, and pHC2.

FIG. 32 is a schematic representation of the heavy chain minilocusconstruct pIGG1 and the κ light chain minilocus construct pKC1, pKVe1,and pKC2.

FIG. 33 depicts a scheme to reconstruct functionally rearranged lightchain genes.

FIG. 34 depicts serum ELISA results

FIG. 35 depicts the results of an ELISA assay of serum from 8 transgenicmice.

FIG. 36 is a schematic representation of plasmid pBCE1.

FIGS. 37A-37C depict the immune response of transgenic mice of thepresent invention against KLH-DNP, by measuring IgG and IgM levelsspecific for KLH-DNP (37A), KLH (37B) and BSA-DNP (37C).

FIG. 38 shows ELISA data demonstrating the presence of antibodies thatbind human carcinoembryonic antigen (CEA) and comprise human μ chains;each panel shows reciprocal serial dilutions from pooled serum samplesobtained from mice on the indicated day following immunization.

FIG. 39 shows ELISA data demonstrating the presence of antibodies thatbind human carcinoembryonic antigen (CEA) and comprise human γ chains;each panel shows reciprocal serial dilutions from pooled serum samplesobtained from mice on the indicated day following immunization.

FIG. 40 shows aligned variable region sequences of 23 randomly-chosencDNAs (SEQ ID NOs. 269, 271, 273, 274, 276-293, 296, 297 and 299-305)generated from mRNA obtained from lymphoid tissue of HC1 transgenic miceimmunized with human carcinoembryonic antigen (CEA) as compared to thegermline transgene sequence (top line) (SEQ ID NO: 269); on each linenucleotide changes relative to germline sequence are shown. The regionscorresponding to heavy chain CDR1, CDR2, and CDR3 are indicated. J chainsequences for J2, J3, J4, J5, and J6 are given as SEQ ID NOs: 207, 272,275, 294 and 298, respectively. Non-germline encoded nucleotides areshown in capital letters.

FIG. 41 shows the nucleotide sequence (SEQ ID NO. 306) of a human DNAfragment, designated vk65.3, containing a V_(κ) gene segment; thededuced amino acid sequences (SEQ ID NO: 307) of the V_(κ) codingregions are also shown; splicing and recombination signal sequences(heptamer/nonamer) are shown boxed.

FIG. 42 shows the nucleotide sequence (SEQ ID NO: 308) of a human DNAfragment, designated vk65.5, containing a V_(κ) gene segment; thededuced amino acid sequences (SEQ ID NO: 309) of the V_(κ) codingregions are also shown; splicing and recombination signal sequences(heptamer/nonamer) are shown boxed.

FIG. 43 shows the nucleotide sequence (SEQ ID NO: ;310) of a human DNAfragment, designated vk65.8, containing a V_(κ) gene segment; thededuced amino acid sequences (SEQ ID NO: 311) of the V_(κ) codingregions are also shown; splicing and recombination signal sequences(heptamer/nonamer) are shown boxed.

FIG. 44 shows the nucleotide sequence (SEQ ID NO: 312) of a human DNAfragment, designated vk65.15, containing a V_(κ) gene segment; thededuced amino acid sequences (SEQ ID NO. 313) of the V_(κ) codingregions are also shown; splicing and recombination signal sequences(heptamer/nonamer) are shown boxed.

FIG. 45 shows formation of a light chain minilocus by homologousrecombination between two overlapping fragments which were co-injected.

FIG. 46 shows ELISA results for monoclonal antibodies reactive with CEAand non-CEA antigens showing the specificity of antigen binding.

FIG. 47 shows the DNA sequences (SEQ ID NO: 314-322) of 10 cDNAsamplified by PCR to amplify transcripts having a human VDJ and a murineconstant region sequence.

FIG. 48 shows ELISA results for various dilutions of serum obtained frommice bearing both a human heavy chain minilocus transgene and a human κminilocus transgene; the mouse was immunized with human CD4 and the datashown represents antibodies reactive with human CD4 and possessing humanκ, human μ, or human γ epitopes, respectively.

FIG. 49 shows relative distribution of lymphocytes staining for human μor mouse μ as determined by FACS for three mouse genotypes.

FIG. 50 shows relative distribution of lymphocytes staining for human κor mouse κ as determined by FACS for three mouse genotypes.

FIG. 51 shows relative distribution of lymphocytes staining for mouse λas determined by FACS for three mouse genotypes.

FIG. 52 shows relative distribution of lymphocytes staining for mouse λor human κ as determined by FACS for four mouse genotypes.

FIG. 53 shows the amounts of human μ, human γ, human κ, mouse μ, mouseγ, mouse κ, and mouse λ chains in the serum of unimmunized 0011 mice.

FIG. 54 shows a scatter plot showing the amounts of human μ, human γ,human κ, mouse μ, mouse γ, mouse κ, and mouse λ chains in the serum ofunimmunized 0011 mice of various genotypes.

FIG. 55 shows the titres of antibodies comprising human μ, human γ, orhuman κ chains in anti-CD4 antibodies in the serum taken at three weeksor seven weeks post-immunization following immunization of a 0011 mousewith human CD4.

FIG. 56 shows a schematic representation of the human heavy chainminilocus transgenes pHC1 and pHC2, and the light chain minilocustransgenes pKC1, pKC1e, and the light chain minilocus transgene createdby homologous recombination between pKC2 and Co4 at the site indicated.

FIG. 57 shows a linkage map of the murine lambda light chain locus astaken from Storb et al. (1989) op.cit.; the stippled boxes represent apseudogene.

FIG. 58 shows a schematic representation of inactivation of the murine λlocus by homologous gene targeting.

FIG. 59 schematically shows the structure of a homologous recombinationtargeting transgene for deleting genes, such as heavy chain constantregion genes.

FIG. 60 shows a map of the BALB/c murine heavy chain locus as taken fromImmunoglobulin Genes, Honjo, T, Alt, F W, and Rabbits T H (eds.)Academic Press, NY (1989) p. 129. Structural genes are shown by closedboxes in the top line; second and third lines show restriction siteswith symbols indicated.

FIG. 61 shows a nucleotide sequence (SEQ ID NO: 323) of mouse heavychain locus a constant region gene.

FIG. 62 shows the construction of a frameshift vector (plasmid B) forintroducing a two bp frameshift into the murine heavy chain locus J₄gene.

FIG. 63 shows isotype specific response of transgenic animals duringhyperimmunization. The relative levels of reactive human μ and γ1 areindicated by a colorimetric ELISA assay (y-axis). We immunized three7-10 week old male HC1 line 57 transgenic animals (#1991, #2356, #2357),in a homozygous JHD background, by intraperitoneal injections of CEA inFreund's adjuvant. The figure depicts binding of 250 fold dilutions ofpooled serum (collected prior to each injection) to CEA coatedmicrotiter wells.

FIGS. 64A and 64B show expression of transgene encoded γ1 isotypemediated by class switch recombination. The genomic structure ofintegrated transgenes in two different human γ1 expressing hybridomas isconsistent with recombination between the μ and γ1 switch regions. FIG.64A shows a Southern blot of PacI/SfiI digested DNA isolated from threetransgene expressing hybridomas. From left to right: clone 92-09A-5H1-5,human γ1⁺/μ⁻; clone 92-90A-4G2-2, human γ1⁺/μ⁻; clone 92-09A-4F7-A5-2,human γ1⁻,μ⁺. All three hybridomas are derived from a 7 month old malemouse hemizygous for the HC1-57 integration, and homozygous for the JHDdisruption (mouse #1991). The blot is hybridized with a probe derivedfrom a 2.3 kb BglII/SfiI DNA fragment spanning the 3′ half of the humanγ1 switch region. No switch product is found in the μ expressinghybridoma, while the two γ1 expressing hybridomas, 92-09A-5H1-5 and92-09A-4G2-2, contain switch products resulting in PacI/SfiI fragmentsof 5.1 and 5.3 kb respectively, FIG. 64B is a diagram of two possibledeletional mechanisms by which a class switch from μ to γ1 can occur.The human μ gene is flanked by 400 bp direct repeats (Σμ and Σμ) whichcan recombine to delete μ. Class switching by this mechanism will alwaysgenerate a 6.4 kb PacI/SfiI fragment, while class switching byrecombination between the μ and the γ1 switch regions will generate aPacI/SfiI fragment between 4 and 7 kb, with size variation betweenindividual switch events. The two γ1 expressing hybridomas examined inFIG. 64A appear to have undergone recombination between the μ and γ1switch regions.

FIG. 65 shows chimeric human/mouse immunoglobulin heavy chains generatedby trans-switching. cDNA clones of trans-switch products were generatedby reverse transcription and PCR amplification of a mixture of spleenand lymph node RNA isolated from a hyperimmunized HC1 transgenic-JHDmouse (#2357; see legend to FIG. 63 for description of animal andimmunization schedule). The partial nucleotide sequence of 10 randomlypicked clones is shown (SEQ ID NOs: 324-332). Lower case lettersindicate germline encoded, capital letters indicate nucleotides thatcannot be assigned to known germline sequences; these may be somaticmutations, N nucleotides, or truncated D segments. Both face typeindicates mouse γ sequences.

FIGS. 66A and 66B show that the rearranged VH251 transgene undergoessomatic mutation in a hyperimmunized. The partial nucleotide sequence ofIgG heavy chain variable region cDNA clones from CH1 line 26 miceexhibiting FIG. 66A primary and FIG. 66B secondary responses to antigen.Germline sequence is shown at the top (SEQ ID NO: 333); nucleotidechanges from germline are given for each clone. A period indicatesidentity with germline sequence, capital letters indicate no identifiedgermline origin. The sequences are grouped according to J segment usage.The germline sequence of each of the J segments is shown (FIG. 66A.2,SEQ ID NOS: 334, 338, 340 and 347; FIG. 66B.2, SEQ ID NOS: 350, 352, 360and 362). Lower case letters within CDR3 sequences indicate identity toknown D segment included in the HC1 transgene. The assigned D segmentsare indicated at the end of each sequence. Unassigned sequences could bederived from N region addition or somatic mutation; or in some casesthey are simply too short to distinguish random N nucleotides from knownD segments. FIG. 66A primary response: 13 randomly picked VH251-γ1 cDNAclones (SEQ ID Nos: 335-337, 339, 341-346, 348 and 349). A 4 week oldfemale HC1 line 26-JHD mouse (#2599) was given a single injection of KLHand complete Freunds adjuvant; spleen cell RNA was isolated 5 dayslater. The overall frequency of somatic mutations within the V segmentis 0.06% (2/3,198 bp). FIG. 66B secondary response: 13 randomly pickedVH251-γ1 cDNA clones (SEQ ID NOs: 351, 353, 355-359, 361 and 363). A 2month old female HC1 line 26-JHD mouse (#3204) was given 3 injections ofHEL and Freunds adjuvant over one month (a primary injection withcomplete adjuvant and boosts with incomplete at one week and 3 weeks);spleen and lymph node RNA was isolated 4 months later. The overallfrequency of somatic mutations within the V segment is 1.6% (52/3,198bp).

FIGS. 67A and 67B show that extensive somatic mutation is confined to γ1sequences: somatic mutation and class switching occur within the samepopulation of B cells. Partial nucleotide sequence of VH251 cDNA clonesisolated from spleen and lymph node cells of HC1 line 57 transgenic-JHDmouse (#2357) hyperimmunized against CEA (see FIG. 63 for immunizationschedule). FIG. 67A: IgM: 23 randomly picked VH251-μ cDNA clones (SEQ IDNO 364). Nucleotide sequence of 156 bp segment including CDRs 1 and 2surrounding residues (SEQ ID NOs.: 364-398). The overall level ofsomatic mutation is 0.1% (5/3,744 bp). FIG. 67B: IgG: 23 randomly pickedVH251-γ1 cDNA clones (SEQ ID NO. 333). Nucleotide sequence of segmentincluding CDRs 1 through 3 and surrounding residues (SEQ ID NO.369-391). J chain sequences for J2, J3, J4, J5 and J6 are given as SEQID NOS. 350, 352, 354, 360 and 362, respectively. The overall frequencyof somatic mutation within the V segment is 1.1% (65/5,658 bp). Forcomparison with the A sequences in FIG. 67A: the mutation frequency forfirst 156 nucleotides is 1.1% (41/3,588 bp). See legend to FIGS. 66A and66B for explanation of symbols.

FIG. 68 indicates that VH51P1 and VH56P1 (SEQ ID NO.: 392), VH51P1 (SEQID NO. 410) and VH4.21 (SEQ ID NO. 412) show extensive somatic mutationof in an unimmunized mouse. The partial nucleotide sequence of IgG heavychain variable region cDNA clones (SEQ ID NOs.: 393-409, 411 and 413)from a 9 week old, unimmunized female HC2 line 2550 transgenic-JHD mouse(#5250). The overall frequency of somatic mutation with the 19 VH56p1segments is 2.2% (101/4,674 bp). The overall frequency of somaticmutation within the single VH5p1 segment is 2.0% (5/246 bp). See legendto FIGS. 66A and 66B for explanation of symbols.

FIG. 69. Double transgenic mice with disrupted endogenous Ig locicontain human IgMκ positive B cells. FACS of cells isolated from spleensof 4 mice with different genotypes. Left column: control mouse (#9944, 6wk old female JH±, JCκ±; heterozygous wild-type mouse heavy and κ-lightchain loci, non-transgenic). Second column: human heavy chain transgenic(#9877, 6 wk old female JH−/−, JCκ−/−, HC2 line 2550+; homozygous fordisrupted mouse heavy and κ-light chain loci, hemizygous for HC2transgene). Third column: human κ-light chain transgenic (#9878, 6 wkold female JH−/−, JCκ−/−, KCo4 line 4437+; homozygous for disruptedmouse heavy and κ-light chain loci, hemizygous for KCo4 transgene).Right column: double transgenic (#9879, 6 wk old female JH−/−m JCκ−/−,HC2 line 2550+, KCo4 line 4437+; homozygous for disrupted mouse heavyand κk-light chain loci, hemizygous for HC2 and KCo4 transgenes). Toprow: spleen cells stained for expression of mouse λ light chain (x-axis)and human κ light chain (y-axis). Second row: spleen cells stained forexpression of human μ heavy chain (x-axis) and human κ light chain(y-axis). Third row: spleen cells stained for expression of mouse pheavy chain (x-axis) and mouse κ light chain (y-axis). Bottom row:histogram of spleen cells stained for expression of mouse B220 antigen(log fluorescence: x-axis; cell number: y-axis). For each of the twocolor panels, the relative number of cells in each of the displayedquadrants is given as percent of a e-parameter gate based on propidiumiodide staining and light scatter. The fraction of B220+ cells in eachof the samples displayed in the bottom row is given as a percent of thelymphocyte light scatter gate.

FIG. 70. Secreted immunoglobulin levels in the serum of doubletransgenic mice. Human μ, γ, and κ, and mouse γ and λ from 18 individualHC2/KCo4 double transgenic mice homozygous for endogenous heavy andK-light chain locus disruption. Mice: (+) HC2 line 2550 (^(˜)5 copies ofHC2 per integration), KCo4 line 4436 (1-2 copies of KCo4 perintegration); (O) HC2 line 2550, KCo4 line 4437 (^(˜)10 copies of KCo4per integration); (x) HC2 line 2550, KCo4 line 4583 (^(˜)5 copies ofKCo4 per integration); (□) HC2 line 2572 (30-50 copies of HC2 perintegration, KCo4 line 4437; (Δ) HC2 line 5467 (20-30 copies of HC2 perintegration, KCo4 line 4437.

FIGS. 71A and 71B show human antibody responses to human antigens. FIG.71A: Primary response to recombinant human soluble CD4. Levels of humanIgM and human κ light chain are reported for prebleed (O) andpost-immunization (●) serum from four double transgenic mice. FIG. 71B:Switching to human IgG occurs in vivo. Human IgG (circles) was detectedwith peroxidase conjugated polyclonal anti-human IgG used in thepresence of 1.5 μ/ml excess IgE, κ and 1% normal mouse serum to inhibitnon-specific cross-reactivity. Human κ light chain (squares) wasdetected using a peroxidase conjugated polyclonal anti-human κ reagentin the presence of 1% normal mouse serum. A representative result fromone mouse (#9344; HC2 line 2550, KCo4 line 4436) is shown. Each pointrepresents an average of duplicate wells minus background absorbance.

FIG. 72 shows FACS analysis of human PBL with a hybridoma supernatantthat discriminates human CD4+ lymphocytes from human CD8+ lymphocytes.

FIG. 73 shows human α-CD4 IgM and IgG in transgenic mouse serum.

FIG. 74 shows competition binding experiments comparing a transgenicmouse α-human CD4 hybridoma monoclonal, 2C11-8, to the RPA-TA and Leu-3Amonoclonals.

FIG. 75 shows production data for Ig expression of cultured 2C11-8hybridoma.

FIG. 76 shows an overlapping set of plasmid inserts constituting theHCo7 transgene.

FIG. 77A depicts the nucleotide sequence (SEQ ID NO: 416) andrestriction map of pGP2b plasmid vector.

FIG. 77B depicts the restriction map of pGP2b plasmid vector.

FIG. 78 (parts A and B) depicts cloning strategy for assembling largetransgenes.

FIG. 79 shows that large inserts are unstable in high-copy pUC derivedplasmids.

FIG. 80 shows phage P1 clone P1-570. Insert spans portion of human heavychain constant region covering γ3 and γ1, together with switch elements.N, NotI; S, SalI, X, XhoI.

FIG. 81 shows serum expression of human μ and γ1 in HCo7 transgenicfounder animals.

FIG. 82 shows serum expression of human immunoglobulins in HCo7/KCo4double transgenic/double deletion mice.

FIG. 83 shows RT PCR detection of human γ1 and γ3 transcripts in HCo7transgenic mouse spleen RNA.

FIG. 84 shows induction of human IgG1 and IgG3 by LPS and IL-4 in vitro.

FIG. 85. Agarose gel electrophoresis apparatus for concentration of YACDNA.

FIG. 86. Two color FACS analysis of bone marrow cells fromHC2/KCo5/JHD/JKD and HC2/KCo4/JHD/JKD mice. The fraction of cells ineach of the B220⁺/CD43⁻, B220⁺/CD43⁺, and B220⁺/IgM⁺ gates is given as apercent.

FIG. 87. Two color FACS analysis of spleen cells from HC2/KCo5/JHD/JKDand HC2/KCo4/JHD/JKD mice. The fraction of cells in each of the B₂₂₀^(bright)/IgM⁺ and B220^(dull)/IgM⁺ gates is given as a percent.

FIG. 88. Binding of IgGκ anti-nCD4 monoclonal antibodies to CD4+ SupT1cells.

FIG. 89 Epitope determination for IgG anti-nCD4 monoclonal antibodies byflow cytometry. SupT1 cells were pre-incubated with buffer (leftcolumn), 2.5 mg/ml RPA-T4 (middle column), or 2.5 mg/ml Leu3a (rightcolumn) and then with one of the 10 human IgG monoclonal antibodies (insupernatant diluted 1:2), or chimeric Leu3a. Results for 3representative human IgG monoclonal antibodies are shown in this figure.

FIG. 90 Inhibition of an MLR by a human IgGk anti-CD4 monoclonalantibody.

FIG. 91 shows the effect of huMAb administration on CD4⁺ cells.

FIG. 92 shows the effect of huMAb administration on CD4⁻ Cells.

FIG. 93 shows the effect of huMAb administration on cynomolgus monkeycells.

FIG. 94 shows the effect of huMAb administration on lymph nodelymphocytes.

FIG. 95 shows serum half-life of huMAbs in cynomolgus monkeys. The dataderived from 1E11 and 6G5 were fit to a two compartment model, whereasthe data derived from 1G2 were fit to a one compartment model.

FIG. 96 shows inhibition by human anti-CD4 mAbs of human cell responsesto tetanus toxoid (TT). Panels (a) and (b) present results for twodifferent assays. Lot numbers of the mAbs are shown in parentheses.

FIG. 97 shows human IgM and IgG anti-IL8 serum titers in transgenicmice. Responses of the individual mice to the immunogen were assessed byELISA, converted to titers (1=OD>0.1 at 1:50 dilution of serum; 2=OD>0.1at 1:250 dilution of serum; 3=OD>0.1 at 1:1250 dilution of serum;4=OD>0.1 at 1:6250 dilution of serum; and 5=OD>0.1 at 1:31250 dilutionof serum) and then averaged.

FIG. 98 shows the effect of human anti-IL8 mAb on IL8-induced neutrophilchemotaxis and elastase release.

Table 1 depicts the sequence of vector pGPe (SEQ ID NO.: 72).

Table 2 depicts the sequence of gene V_(H)49.8 (SEQ ID NO.: 79-80).

Table 3 depicts the detection of human IgM and IgG in the serum oftransgenic mice of this invention.

Table 4 depicts sequences of VDJ joints.

Table 5 depicts the distribution of J segments incorporated into pHC1transgene encoded transcripts to J segments found in adult humanperipheral blood lymphocytes (PBL).

Table 6 depicts the distribution of D segments incorporated into pHC1transgene encoded transcripts to D segments found in adult humanperipheral blood lymphocytes (PBL).

Table 7 depicts the length of the CDR3 peptides from transcripts within-frame VDJ joints (SEQ ID NOs.116-145). in the pHC1 transgenic mouseand in human PBL.

Table 8 depicts the predicted amino acid sequences of the VDJ regionsfrom 30 clones analyzed from a pHC1 transgenic.

Table 9 shows transgenic mice of line 112 that were used in theindicated experiments; (+) indicates the presence of the respectivetransgene, (++) indicates that the animal is homozygous for the J_(H)Dknockout transgene.

Table 10 shows the genotypes of several 0011 mice.

Table 11 shows human variable region usage in hybridomas from transgenicmice.

Table 12 shows transgene V and J segment usage.

Table 13 shows the occurrence of somatic mutation in the HC2 heavy chaintransgene in transgenic mice.

Table 14 shows identification of human V_(κ) segments on the YAC 4x17E1.

Table 15. Identification of human vk genes expressed in mouse lineKCo5-9272.

Table 16. Secretion levels for human IgGk Anti-nCD4 monoclonalantibodies

Table 17. Rate and affinity constants for monoclonal antibodies thatbind to human CD4.

Table 18. Affinity and rate constants of human anti-human CD4 monoclonalantibodies.

Table 19. Avidity and rate constants of human anti-human CD4 monoclonalantibodies.

Table 20. Avidity and rate constants reported for anti CD4 monoclonalantibodies.

Table 21. Avidity constants of human anti-human CD4 monoclonalantibodies as determined by flow cytometry.

Table 22. Partial Nucleotide Sequence for Functional Transcripts.

Table 23 Germline V(D)J Segment Usage in Hybridoma Transcripts.

Table 24. Primers, Vectors and Products Used in Minigene Construction.

Table 25. Effect of Human mAbs on Peripheral Chimpanzee Lymphocytes.

Table 26. Summary of Flow Cytometry Studies on Lymph Node Lymphocytes.

Table 27. Monoclonal Antibody Secretion, Avidity and Rate Constants.

Table 28. Specificity and Characterization of Human Anti-IL8 mAbs.

DETAILED DESCRIPTION

As has been discussed supra, it is desirable to produce humanimmunoglobulins that are reactive with specific human antigens that arepromising therapeutic and/or diagnostic targets. However, producinghuman immunoglobulins that bind specifically with human antigens isproblematic.

First, the immunized animal that serves as the source of B cells mustmake an immune response against the presented antigen. In order for ananimal to make an immune response, the antigen presented must be foreignand the animal must not be tolerant to the antigen. Thus, for example,if it is desired to produce a human monoclonal antibody with an idiotypethat binds to a human protein, self-tolerance will prevent an immunizedhuman from making a substantial immune response to the human protein,since the only epitopes of the antigen that may be immunogenic will bethose that result from polymorphism of the protein within the humanpopulation (allogeneic epitopes).

Second, if the animal that serves as the source of B-cells for forming ahybridoma (a human in the illustrative given example) does make animmune response against an authentic self antigen, a severe autoimmunedisease may result in the animal. Where humans would be used as a sourceof B-cells for a hybridoma, such autoimmunization would be consideredunethical by contemporary standards. Thus, developing hybridomassecreting human immunoglobulin chains specifically reactive withpredetermined human antigens is problematic, since a reliable source ofhuman antibody-secreting B cells that can evoke an antibody responseagainst predetermined human antigens is needed.

One methodology that can be used to obtain human antibodies that arespecifically reactive with human antigens is the production of atransgenic mouse harboring the human immunoglobulin transgene constructsof this invention. Briefly, transgenes containing all or portions of thehuman immunoglobulin heavy and light chain loci, or transgenescontaining synthetic “miniloci” (described infra, and in copendingapplications U.S. Ser. No. 08/352,322, filed 7 Dec. 1994, U.S. Ser. No.07/990,860, filed 16 Dec. 1992, U.S. Ser. No. 07/810,279 filed 17 Dec.1991, U.S. Ser. No. 07/904,068 filed 23 Jun. 1992; U.S. Ser. No.07/853,408, filed 18 Mar. 1992, U.S. Ser. No. 07/574,748 filed Aug. 29,1990, U.S. Ser. No. 07/575,962 filed Aug. 31, 1990, and PCT/US91/06185filed Aug. 28, 1991, each incorporated herein by reference) whichcomprise essential functional elements of the human heavy and lightchain loci, are employed to produce a transgenic nonhuman animal. Such atransgenic nonhuman animal will have the capacity to produceimmunoglobulin chains that are encoded by human immunoglobulin genes,and additionally will be capable of making an immune response againsthuman antigens. Thus, such transgenic animals can serve as a source ofimmune sera reactive with specified human antigens, and B-cells fromsuch transgenic animals can be fused with myeloma cells to producehybridomas that secrete monoclonal antibodies that are encoded by humanimmunoglobulin genes and which are specifically reactive with humanantigens.

The production of transgenic mice containing various forms ofimmunoglobulin genes has been reported previously. Rearranged mouseimmunoglobulin heavy or light chain genes have been used to producetransgenic mice. In addition, functionally rearranged human Ig genesincluding the μ or γ1 constant region have been expressed in transgenicmice. However, experiments in which the transgene comprises unrearranged(V-D-J or V-J not rearranged) immunoglobulin genes have been variable,in some cases, producing incomplete or minimal rearrangement of thetransgene. However, there are no published examples of either rearrangedor unrearranged immunoglobulin transgenes which undergo successfulisotype switching between C_(H) genes within a transgene.

The invention also provides a method for identifying candidatehybridomas which secrete a monoclonal antibody comprising a humanimmunoglobulin chain consisting essentially of a human VDJ sequence inpolypeptide linkage to a human constant region sequence. Such candidatehybridomas are identified from a pool of hybridoma clones comprising:(1) hybridoma clones that express immunoglobulin chains consistingessentially of a human VDJ region and a human constant region, and (2)trans-switched hybridomas that express heterohybrid immunoglobulinchains consisting essentially of a human VDJ region and a murineconstant region. The supernatant(s) of individual or pooled hybridomaclones is contacted with a predetermined antigen, typically an antigenwhich is immobilized by adsorption onto a solid substrate (e.g., amicrotitre well), under binding conditions to select antibodies havingthe predetermined antigen binding specificity. An antibody thatspecifically binds to human constant regions is also contacted with thehybridoma supernatant and predetermined antigen under binding conditionsso that the antibody selectively binds to at least one human constantregion epitope but substantially does not bind to murine constant regionepitopes; thus forming complexes consisting essentially of hybridomasupernatant (transgenic monoclonal antibody) bound to a predeterminedantigen and to an antibody that specifically binds human constantregions (and which may be labeled with a detectable label or reporter).Detection of the formation of such complexes indicates hybridoma clonesor pools which express a human immunoglobulin chain.

In a preferred embodiment of the invention, the anti-human constantregion immunoglobulin used in screening specifically recognizes a non-μ,non-δ isotype, preferably a α or ε, more preferably a γ isotype constantregion. Monoclonal antibodies of the γ isotype are preferred (i) becausethe characteristics of IgG immunoglobulins are preferable to IgMimmunogloblins for some therapeutic applications (e.g., due to thesmaller size of the IgG dimers compared to IgM pentamers) and, (ii)because the process of somatic mutation is correlated with the classswitch from the μ constant region to the non-μ (e.g., γ) constantregions. Immunoglobulins selected from the population of immunoglobulinsthat have undergone class switch (e.g., IgG) tend to bind antigen withhigher affinity than immunoglobulins selected from the population thathas not undergone class switch (e.g., IgM). See, e.g., Lonberg andHuszar. Intern. Rev. Immunol. 13:65-93 (1995) which is incorporatedherein by reference.

In one embodiment the candidate hybridomas are first screened for the γisotype constant region and the pool of IgG-expressing hybridomas isthen screened for specific binding to the predetermined antigen.

Thus, according to the method, a transgenic mouse of the invention isimmunized with the predetermined antigen to induce an immune response. Bcells are collected from the mouse and fused to immortal cells toproduce hybridomas. The hybridomas are first screened to identifyindividual hybridomas secreting Ig of a non-mu, non-delta isotype (e.g.,IgG). This set of hybridomas is then screened for specific binding tothe predetermined antigen of interest. Screening is carried out usingstandard techniques as described in, e.g., Harlow and Lane, Antibodies:A Laboratory Manual, Cold Spring Harbor, N.Y. (1988). Using this methodit is possible to identify high-affinity immunoglobulins (e.g., Kagreater than about 10⁷ M⁻¹) practically and efficiently.

Definitions

As used herein, the term “antibody” refers to a glycoprotein comprisingat least two light polypeptide chains and two heavy polypeptide chains.Each of the heavy and light polypeptide chains contains a variableregion (generally the amino terminal portion of the polypeptide chain)which contains a binding domain which interacts with antigen. Each ofthe heavy and light polypeptide chains also comprises a constant regionof the polypeptide chains (generally the carboxyl terminal portion)which may mediate the binding of the immunoglobulin to host tissues orfactors including various cells of the immune system, some phagocyticcells and the first component (C1q) of the classical complement system.

As used herein, a “heterologous antibody” is defined in relation to thetransgenic non-human organism producing such an antibody. It is definedas an antibody having an amino acid sequence or an encoding DNA sequencecorresponding to that found in an organism not consisting of thetransgenic non-human animal, and generally from a species other thanthat of the transgenic non-human animal.

As used herein, a “heterohybrid antibody” refers to an antibody having alight and heavy chains of different organismal origins. For example, anantibody having a human heavy chain associated with a murine light chainis a heterohybrid antibody.

As used herein, “isotype” refers to the antibody class (e.g., IgM orIgG₁) that is encoded by heavy chain constant region genes.

As used herein, “isotype switching” refers to the phenomenon by whichthe class, or isotype, of an antibody changes from one Ig class to oneof the other Ig classes.

As used herein, “nonswitched isotype” refers to the isotypic class ofheavy chain that is produced when no isotype switching has taken place;the C_(H) gene encoding the nonswitched isotype is typically the firstC_(H) gene immediately downstream from the functionally rearranged VDJgene.

As used herein, the term “switch sequence” refers to those DNA sequencesresponsible for switch recombination. A “switch donor” sequence,typically a μ switch region, will be 5′ (i.e., upstream) of theconstruct region to be deleted during the switch recombination. The“switch acceptor” region will be between the construct region to bedeleted and the replacement constant region (e.g., γ, ε, etc.). As thereis no specific site where recombination always occurs, the final genesequence will typically not be predictable from the construct.

As used herein, “glycosylation pattern” is defined as the pattern ofcarbohydrate units that are covalently attached to a protein, morespecifically to an immunoglobulin protein. A glycosylation pattern of aheterologous antibody can be characterized as being substantiallysimilar to glycosylation patterns which occur naturally on antibodiesproduced by the species of the nonhuman transgenic animal, when one ofordinary skill in the art would recognize the glycosylation pattern ofthe heterologous antibody as being more similar to said pattern ofglycosylation in the species of the nonhuman transgenic animal than tothe species from which the C_(H) genes of the transgene were derived.

As used herein, “specific binding” refers to the property of theantibody: (1) to bind to a predetermined antigen with an affinity of atleast 1×10⁷ M⁻¹, and (2) to preferentially bind to the predeterminedantigen with an affinity that is at least two-fold greater than itsaffinity for binding to a non-specific antigen (e.g., BSA, casein) otherthan the predetermined antigen or a closely-related antigen.

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally-occurring.

The term “rearranged” as used herein refers to a configuration of aheavy chain or light chain immunoglobulin locus wherein a V segment ispositioned immediately adjacent to a D-J or J segment in a conformationencoding essentially a complete V_(H) or V_(L) domain, respectively. Arearranged immunoglobulin gene locus can be identified by comparison togermline DNA; a rearranged locus will have at least one recombinedheptamer/nonamer homology element.

The term “unrearranged” or “germline configuration” as used herein inreference to a V segment refers to the configuration wherein the Vsegment is not recombined so as to be immediately adjacent to a D or Jsegment.

For nucleic acids, the term “substantial homology” indicates that twonucleic acids, or designated sequences thereof, when optimally alignedand compared, are identical, with appropriate nucleotide insertions ordeletions, in at least about 80% of the nucleotides, usually at leastabout 90% to 95%, and more preferably at least about 98 to 99.5% of thenucleotides. Alternatively, substantial homology exists when thesegments will hybridize under selective hybridization conditions, to thecomplement of the strand. The nucleic acids may be present in wholecells, in a cell lysate, or in a partially purified or substantiallypure form. A nucleic acid is “isolated” or “rendered substantially pure”when purified away from other cellular components or other contaminants,e.g., other cellular nucleic acids or proteins, by standard techniques,including alkaline/SDS treatment, CsCl banding, column chromatography,agarose gel electrophoresis and others well known in the art. See, F.Ausubel, et al., ed. Current Protocols in Molecular Biology, GreenePublishing and Wiley-Interscience, New York (1987).

The nucleic acid compositions of the present invention, while often in anative sequence (except for modified restriction sites and the like),from either cDNA, genomic or mixtures may be mutated, thereof inaccordance with standard techniques to provide gene sequences. Forcoding sequences, these mutations, may affect amino acid sequence asdesired. In particular, DNA sequences substantially homologous to orderived from native V, D, J, constant, switches and other such sequencesdescribed herein are contemplated (where “derived” indicates that asequence is identical or modified from another sequence).

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For instance, apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the sequence. With respect to transcriptionregulatory sequences, operably linked means that the DNA sequences beinglinked are contiguous and, where necessary to join two protein codingregions, contiguous and in reading frame. For switch sequences, operablylinked indicates that the sequences are capable of effecting switchrecombination.

Transgenic Nonhuman Animals Capable of Producing Heterologous Antibodies

The design of a transgenic non-human animal that responds to foreignantigen stimulation with a heterologous antibody repertoire, requiresthat the heterologous immunoglobulin transgenes contained within thetransgenic animal function correctly throughout the pathway of B-celldevelopment. In a preferred embodiment, correct function of aheterologous heavy chain transgene includes isotype switching.Accordingly, the transgenes of the invention are constructed so as toproduce isotype switching and one or more of the following: (1) highlevel and cell-type specific expression, (2) functional generearrangement, (3) activation of and response to allelic exclusion, (4)expression of a sufficient primary repertoire, (5) signal transduction,(6) somatic hypermutation, and (7) domination of the transgene antibodylocus during the immune response.

As will be apparent from the following disclosure, not all of theforegoing criteria need be met. For example, in those embodimentswherein the endogenous immunoglobulin loci of the transgenic animal arefunctionally disrupted, the transgene need not activate allelicexclusion. Further, in those embodiments wherein the transgene comprisesa functionally rearranged heavy and/or light chain immunoglobulin gene,the second criteria of functional gene rearrangement is unnecessary, atleast for that transgene which is already rearranged. For background onmolecular immunology, see, Fundamental Immunology, 2nd edition (1989),Paul William E., ed. Raven Press, N.Y., which is incorporated herein byreference.

In one aspect of the invention, transgenic non-human animals areprovided that contain rearranged, unrearranged or a combination ofrearranged and unrearranged heterologous immunoglobulin heavy and lightchain transgenes in the germline of the transgenic animal. Each of theheavy chain transgenes comprises at least one C_(H) gene. In addition,the heavy chain transgene may contain functional isotype switchsequences, which are capable of supporting isotype switching of aheterologous transgene encoding multiple C_(H) genes in B-cells of thetransgenic animal. Such switch sequences may be those which occurnaturally in the germline immunoglobulin locus from the species thatserves as the source of the transgene C_(H) genes, or such switchsequences may be derived from those which occur in the species that isto receive the transgene construct (the transgenic animal). For example,a human transgene construct that is used to produce a transgenic mousemay produce a higher frequency of isotype switching events if itincorporates switch sequences similar to those that occur naturally inthe mouse heavy chain locus, as presumably the mouse switch sequencesare optimized to function with the mouse switch recombinase enzymesystem, whereas the human switch sequences are not. Switch sequencesmade be isolated and cloned by conventional cloning methods, or may besynthesized de novo from overlapping synthetic oligonucleotides designedon the basis of published sequence information relating toimmunoglobulin switch region sequences (Mills et al., Nucl. Acids Res.18:7305-7316 (1991); Sideras et al., Intl. Immunol. 1:631-642 (1989),which are incorporated herein by reference).

For each of the foregoing transgenic animals, functionally rearrangedheterologous heavy and light chain immunoglobulin transgenes are foundin a significant fraction of the B-cells of the transgenic animal (atleast 10 percent).

The transgenes of the invention include a heavy chain transgenecomprising DNA encoding at least one variable gene segment, onediversity gene segment, one joining gene segment and at least oneconstant region gene segment. The immunoglobulin light chain transgenecomprises DNA encoding at least one variable gene segment, one joininggene segment and at least one constant region gene segment. The genesegments encoding the light and heavy chain gene segments areheterologous to the transgenic non-human animal in that they are derivedfrom, or correspond to, DNA encoding immunoglobulin heavy and lightchain gene segments from a species not consisting of the transgenicnon-human animal. In one aspect of the invention, the transgene isconstructed such that the individual gene segments are unrearranged,i.e., not rearranged so as to encode a functional immunoglobulin lightor heavy chain. Such unrearranged transgenes support recombination ofthe V, D, and J gene segments (functional rearrangement) and preferablysupport incorporation of all or a portion of a D region gene segment inthe resultant rearranged immunoglobulin heavy chain within thetransgenic non-human animal when exposed to antigen.

In an alternate embodiment, the transgenes comprise an unrearranged“mini-locus”. Such transgenes typically comprise a substantial portionof the C, D, and J segments as well as a subset of the V gene segments.In such transgene constructs, the various regulatory sequences, e.g.promoters, enhancers, class switch regions, splice-donor andsplice-acceptor sequences for RNA processing, recombination signals andthe like, comprise corresponding sequences derived from the heterologousDNA. Such regulatory sequences may be incorporated into the transgenefrom the same or a related species of the non-human animal used in theinvention. For example, human immunoglobulin gene segments may becombined in a transgene with a rodent immunoglobulin enhancer sequencefor use in a transgenic mouse. Alternatively, synthetic regulatorysequences may be incorporated into the transgene, wherein such syntheticregulatory sequences are not homologous to a functional DNA sequencethat is known to occur naturally in the genomes of mammals. Syntheticregulatory sequences are designed according to consensus rules, such as,for example, those specifying the permissible sequences of asplice-acceptor site or a promoter/enhancer motif. For example, aminilocus comprises a portion of the genomic immunoglobulin locus havingat least one internal (i.e., not at a terminus of the portion) deletionof a non-essential DNA portion (e.g., intervening sequence; intron orportion thereof) as compared to the naturally-occurring germline Iglocus.

The invention also includes transgenic animals containing germ linecells having a heavy and light transgene wherein one of the saidtransgenes contains rearranged gene segments with the other containingunrearranged gene segments. In the preferred embodiments, the rearrangedtransgene is a light chain immunoglobulin transgene and the unrearrangedtransgene is a heavy chain immunoglobulin transgene.

The Structure and Generation of Antibodies

The basic structure of all immunoglobulins is based upon a unitconsisting of two light polypeptide chains and two heavy polypeptidechains. Each light chain comprises two regions known as the variablelight chain region and the constant light chain region. Similarly, theimmunoglobulin heavy chain comprises two regions designated the variableheavy chain region and the constant heavy chain region.

The constant region for the heavy or light chain is encoded by genomicsequences referred to as heavy or light constant region gene (C_(H))segments. The use of a particular heavy chain gene segment defines theclass of immunoglobulin. For example, in humans, the μ constant regiongene segments define the IgM class of antibody whereas the use of a γ,γ2, γ3 or γ4 constant region gene segment defines the IgG class ofantibodies as well as the IgG subclasses IgG1 through IgG4. Similarly,the use of a α₁ or α₂ constant region gene segment defines the IgA classof antibodies as well as the subclasses IgA1 and IgA2. The δ and εconstant region gene segments define the IgD and IgE antibody classes,respectively.

The variable regions of the heavy and light immunoglobulin chainstogether contain the antigen binding domain of the antibody. Because ofthe need for diversity in this region of the antibody to permit bindingto a wide range of antigens, the DNA encoding the initial or primaryrepertoire variable region comprises a number of different DNA segmentsderived from families of specific variable region gene segments. In thecase of the light chain variable region, such families comprise variable(V) gene segments and joining (J) gene segments. Thus, the initialvariable region of the light chain is encoded by one V gene segment andone J gene segment each selected from the family of V and J genesegments contained in the genomic DNA of the organism. In the case ofthe heavy chain variable region, the DNA encoding the initial or primaryrepertoire variable region of the heavy chain comprises one heavy chainV gene segment, one heavy chain diversity (D) gene segment and one Jgene segment, each selected from the appropriate V, D and J families ofimmunoglobulin gene segments in genomic DNA.

In order to increase the diversity of sequences that contribute toforming antibody binding sites, it is preferable that a heavy chaintransgene include cis-acting sequences that support functional V-D-Jrearrangement that can incorporate all or part of a D region genesequence in a rearranged V-D-J gene sequence. Typically, at least about1 percent of expressed transgene-encoded heavy chains (or mRNAs) includerecognizable D region sequences in the V region. Preferably, at leastabout 10 percent of transgene-encoded V regions include recognizable Dregion sequences, more preferably at least about 30 percent, and mostpreferably more than 50 percent include recognizable D region sequences.

A recognizable D region sequence is generally at least about eightconsecutive nucleotides corresponding to a sequence present in a Dregion gene segment of a heavy chain transgene and/or the amino acidsequence encoded by such D region nucleotide sequence. For example, if atransgene includes the D region gene DHQ52, a transgene-encoded mRNAcontaining the sequence 5′-TAACTGGG-3′ located in the V region between aV gene segment sequence and a J gene segment sequence is recognizable ascontaining a D region sequence, specifically a DHQ52 sequence.Similarly, for example, if a transgene includes the D region gene DHQ52,a transgene-encoded heavy chain polypeptide containing the amino acidsequence -DAF- located in the V region between a V gene segment aminoacid sequence and a J gene segment amino acid sequence may berecognizable as containing a D region sequence, specifically a DHQ52sequence. However, since D region segments may be incorporated in VDJjoining to various extents and in various reading frames, a comparisonof the D region area of a heavy chain variable region to the D regionsegments present in the transgene is necessary to determine theincorporation of particular D segments. Moreover, potential exonucleasedigestion during recombination may lead to imprecise V-D and D-J jointsduring V-D-J recombination.

However, because of somatic mutation and N-region addition, some Dregion sequences may be recognizable but may not correspond identicallyto a consecutive D region sequence in the transgene. For example, anucleotide sequence 5′-CTAAXTGGGG-3′ (SEQ ID NO.: 1)., where X is A, T,or G, and which is located in a heavy chain V region and flanked by a Vregion gene sequence and a J region gene sequence, can be recognized ascorresponding to the DHQ52 sequence 5′-CTAACTGGG-3.′. Similarly, forexample, the polypeptide sequences -DAFDI-(SEQ ID NO.: 2), -DYFDY-(SEQID NO.: 3)-, or -GAFDI-(SEQ ID NO.: 4) located in a V region and flankedon the amino-terminal side by an amino acid sequence encoded by atransgene V gene sequence and flanked on the carboxy terminal side by anamino acid sequence encoded by a transgene J gene sequence isrecognizable as a D region sequence.

Therefore, because somatic mutation and N-region addition can producemutations in sequences derived from a transgene D region, the followingdefinition is provided as a guide for determining the presence of arecognizable D region sequence. An amino acid sequence or nucleotidesequence is recognizable as a D region sequence if: (1) the sequence islocated in a V region and is flanked on one side by a V gene sequence(nucleotide sequence or deduced amino acid sequence) and on the otherside by a J gene sequence (nucleotide sequence or deduced amino acidsequence) and (2) the sequence is substantially identical orsubstantially similar to a known D gene sequence (nucleotide sequence orencoded amino acid sequence).

The term “substantial identity” as used herein denotes a characteristicof a polypeptide sequence or nucleic acid sequence, wherein thepolypeptide sequence has at least 50 percent sequence identity comparedto a reference sequence, often at least about 80% sequence identity andsometimes more than about 90% sequence identity, and the nucleic acidsequence has at least 70 percent sequence identity compared to areference sequence. The percentage of sequence identity is calculatedexcluding small deletions or additions which total less than 35 percentof the reference sequence. The reference sequence may be a subset of alarger sequence, such as an entire D gene; however, the referencesequence is at least 8 nucleotides long in the case of polynucleotides,and at least 3 amino residues long in the case of a polypeptide.Typically, the reference sequence is at least 8 to 12 nucleotides or atleast 3 to 4 amino acids, and preferably the reference sequence is 12 to15 nucleotides or more, or at least 5 amino acids.

The term “substantial similarity” denotes a characteristic of anpolypeptide sequence, wherein the polypeptide sequence has at least 80percent similarity to a reference sequence. The percentage of sequencesimilarity is calculated by scoring identical amino acids or positionalconservative amino acid substitutions as similar. A positionalconservative amino acid substitution is one that can result from asingle nucleotide substitution; a first amino acid is replaced by asecond amino acid where a codon for the first amino acid and a codon forthe second amino acid can differ by a single nucleotide substitution.Thus, for example, the sequence -Lys-Glu-Arg-Val-(SEQ ID NO.: 5) issubstantially similar to the sequence -Asn-Asp-Ser-Val-(SEQ ID NO.: 6),since the codon sequence -AAA-GAA-AGA-GUU-(SEQ ID NO.: 7) can be mutatedto -AAC-GAC-AGC-GUU-(SEQ ID NO.:8) by introducing only 3 substitutionmutations, single nucleotide substitutions in three of the four originalcodons. The reference sequence may be a subset of a larger sequence,such as an entire D gene; however, the reference sequence is at least 4amino residues long. Typically, the reference sequence is at least 5amino acids, and preferably the reference sequence is 6 amino acids ormore.

The Primary Repertoire

The process for generating DNA encoding the heavy and light chainimmunoglobulin genes occurs primarily in developing B-cells. Prior tothe joining of various immunoglobulin gene segments, the V, D, J andconstant (C) gene segments are found, for the most part, in clusters ofV, D, J and C gene segments in the precursors of primary repertoireB-cells. Generally, all of the gene segments for a heavy or light chainare located in relatively close proximity on a single chromosome. Suchgenomic DNA prior to recombination of the various immunoglobulin genesegments is referred to herein as “unrearranged” genomic DNA. DuringB-cell differentiation, one of each of the appropriate family members ofthe V, D, J (or only V and J in the case of light chain genes) genesegments are recombined to form functionally rearranged heavy and lightimmunoglobulin genes. Such functional rearrangement is of the variableregion segments to form DNA encoding a functional variable region. Thisgene segment rearrangement process appears to be sequential. First,heavy chain D-to-J joints are made, followed by heavy chain V-to-DJjoints and light chain V-to-J joints. The DNA encoding this initial formof a functional variable region in a light and/or heavy chain isreferred to as “functionally rearranged DNA” or “rearranged DNA”. In thecase of the heavy chain, such DNA is referred to as “rearranged heavychain DNA” and in the case of the light chain, such DNA is referred toas “rearranged light chain DNA”. Similar language is used to describethe functional rearrangement of the transgenes of the invention.

The recombination of variable region gene segments to form functionalheavy and light chain variable regions is mediated by recombinationsignal sequences (RSS's) that flank recombinationally competent V, D andJ segments. RSS's necessary and sufficient to direct recombination,comprise a dyad-symmetric heptamer, an AT-rich nonamer and anintervening spacer region of either 12 or 23 base pairs. These signalsare conserved among the different loci and species that carry out D-J(or V-J) recombination and are functionally interchangeable. SeeOettinger, et al. (1990), Science, 248, 1517-1523 and references citedtherein. The heptamer comprises the sequence CACAGTG or its analoguefollowed by a spacer of unconserved sequence and then a nonamer havingthe sequence ACAAAAACC or its analogue. These sequences are found on theJ, or downstream side, of each V and D gene segment. Immediatelypreceding the germline D and J segments are again two recombinationsignal sequences, first the nonamer and then the heptamer againseparated by an unconserved sequence. The heptameric and nonamericsequences following a V_(L), V_(H) or D segment are complementary tothose preceding the J_(L), D or J_(H) segments with which theyrecombine. The spacers between the heptameric and nonameric sequencesare either 12 base pairs long or between 22 and 24 base pairs long.

In addition to the rearrangement of V, D and J segments, furtherdiversity is generated in the primary repertoire of immunoglobulin heavyand light chain by way of variable recombination between the V and Jsegments in the light chain and between the D and J segments of theheavy chain. Such variable recombination is generated by variation inthe exact place at which such segments are joined. Such variation in thelight chain typically occurs within the last codon of the V gene segmentand the first codon of the J segment. Similar imprecision in joiningoccurs on the heavy chain chromosome between the D and J_(H) segmentsand may extend over as many as 10 nucleotides. Furthermore, severalnucleotides may be inserted between the D and J_(H) and between theV_(H) and D gene segments which are not encoded by genomic DNA. Theaddition of these nucleotides is known as N-region diversity.

After VJ and/or VDJ rearrangement, transcription of the rearrangedvariable region and one or more constant region gene segments locateddownstream from the rearranged variable region produces a primary RNAtranscript which upon appropriate RNA splicing results in an mRNA whichencodes a full length heavy or light immunoglobulin chain. Such heavyand light chains include a leader signal sequence to effect secretionthrough and/or insertion of the immunoglobulin into the transmembraneregion of the B-cell. The DNA encoding this signal sequence is containedwithin the first exon of the V segment used to form the variable regionof the heavy or light immunoglobulin chain. Appropriate regulatorysequences are also present in the mRNA to control translation of themRNA to produce the encoded heavy and light immunoglobulin polypeptideswhich upon proper association with each other form an antibody molecule.

The net effect of such rearrangements in the variable region genesegments and the variable recombination which may occur during suchjoining, is the production of a primary antibody repertoire. Generally,each B-cell which has differentiated to this stage, produces a singleprimary repertoire antibody. During this differentiation process,cellular events occur which suppress the functional rearrangement ofgene segments other than those contained within the functionallyrearranged Ig gene. The process by which diploid B-cells maintain suchmono-specificity is termed allelic exclusion.

The Secondary Repertoire

B-cell clones expressing immunoglobulins from within the set ofsequences comprising the primary repertoire are immediately available torespond to foreign antigens. Because of the limited diversity generatedby simple VJ and VDJ joining, the antibodies produced by the so-calledprimary response are of relatively low affinity. Two different types ofB-cells make up this initial response: precursors of primaryantibody-forming cells and precursors of secondary repertoire B-cells(Linton et al., Cell 59:1049-1059 (1989)). The first type of B-cellmatures into IgM-secreting plasma cells in response to certain antigens.The other B-cells respond to initial exposure to antigen by entering aT-cell dependent maturation pathway.

During the T-cell dependent maturation of antigen stimulated B-cellclones, the structure of the antibody molecule on the cell surfacechanges in two ways: the constant region switches to a non-IgM subtypeand the sequence of the variable region can be modified by multiplesingle amino acid substitutions to produce a higher affinity antibodymolecule.

As previously indicated, each variable region of a heavy or light Igchain contains an antigen binding domain. It has been determined byamino acid and nucleic acid sequencing that somatic mutation during thesecondary-response occurs throughout the V region including the threecomplementary determining regions (CDR1, CDR2 and CDR3) also referred toas hypervariable regions 1, 2 and 3 (Kabat et al. Sequences of Proteinsof Immunological Interest (1991) U.S. Department of Health and HumanServices, Washington, D.C., incorporated herein by reference. The CDR1and CDR2 are located within the variable gene segment whereas the CDR3is largely the result of recombination between V and J gene segments orV, D and J gene segments. Those portions of the variable region which donot consist of CDR1, 2 or 3 are commonly referred to as frameworkregions designated FR1, FR2, FR3 and FR4. See FIG. 1. Duringhypermutation, the rearranged DNA is mutated to give rise to new cloneswith altered Ig molecules. Those clones with higher affinities for theforeign antigen are selectively expanded by helper T-cells, giving riseto affinity maturation of the expressed antibody. Clonal selectiontypically results in expression of clones containing new mutation withinthe CDR1, 2 and/or 3 regions. However, mutations outside these regionsalso occur which influence the specificity and affinity of the antigenbinding domain.

Transgenic Non-Human Animals Capable of Producing Heterologous Antibody

Transgenic non-human animals in one aspect of the invention are producedby introducing at least one of the immunoglobulin transgenes of theinvention (discussed hereinafter) into a zygote or early embryo of anon-human animal. The non-human animals which are used in the inventiongenerally comprise any mammal which is capable of rearrangingimmunoglobulin gene segments to produce a primary antibody response.Such nonhuman transgenic animals may include, for example,transgenic-pigs, transgenic rats, transgenic rabbits, transgenic cattle,and other transgenic animal species, particularly-mammalian species,known in the art. A particularly preferred non-human animal is the mouseor other members of the rodent family.

However, the invention is not limited to the use of mice. Rather, anynon-human mammal which is capable of mounting a primary and secondaryantibody response may be used. Such animals include non-human primates,such as chimpanzee, bovine, ovine, and porcine species, other members ofthe rodent family, e.g. rat, as well as rabbit and guinea pig.Particular preferred animals are mouse, rat, rabbit and guinea pig, mostpreferably mouse.

In one embodiment of the invention, various gene segments from the humangenome are used in heavy and light chain transgenes in an unrearrangedform. In this embodiment, such transgenes are introduced into mice. Theunrearranged gene segments of the light and/or heavy chain transgenehave DNA sequences unique to the human species which are distinguishablefrom the endogenous immunoglobulin gene segments in the mouse genome.They may be readily detected in unrearranged form in the germ line andsomatic cells not consisting of B-cells and in rearranged form inB-cells.

In an alternate embodiment of the invention, the transgenes compriserearranged heavy and/or light immunoglobulin transgenes. Specificsegments of such transgenes corresponding to functionally rearranged VDJor VJ segments, contain immunoglobulin DNA sequences which are alsoclearly distinguishable from the endogenous immunoglobulin gene segmentsin the mouse.

Such differences in DNA sequence are also reflected in the amino acidsequence encoded by such human immunoglobulin transgenes as compared tothose encoded by mouse B-cells. Thus, human immunoglobulin amino acidsequences may be detected in the transgenic non-human animals of theinvention with antibodies specific for immunoglobulin epitopes encodedby human immunoglobulin gene segments.

Transgenic B-cells containing unrearranged transgenes from human orother species functionally recombine the appropriate gene segments toform functionally rearranged light and heavy chain variable regions. Itwill be readily apparent that the antibody encoded by such rearrangedtransgenes has a DNA and/or amino acid sequence which is heterologous tothat normally encountered in the nonhuman animal used to practice theinvention.

Unrearranged Transgenes

As used herein, an “unrearranged immunoglobulin heavy chain transgene”comprises DNA encoding at least one variable gene segment, one diversitygene segment, one joining gene segment and one constant region genesegment. Each of the gene segments of said heavy chain transgene arederived from, or has a sequence corresponding to, DNA encodingimmunoglobulin heavy chain gene segments from a species not consistingof the non-human animal into which said transgene is introduced.Similarly, as used herein, an “unrearranged immunoglobulin light chaintransgene” comprises DNA encoding at least one variable gene segment,one joining gene segment and at least one constant region gene segmentwherein each gene segment of said light chain transgene is derived from,or has a sequence corresponding to, DNA encoding immunoglobulin lightchain gene segments from a species not consisting of the non-humananimal into which said light chain transgene is introduced.

Such heavy and light chain transgenes in this aspect of the inventioncontain the above-identified gene segments in an unrearranged form.Thus, interposed between the V, D and J segments in the heavy chaintransgene and between the V and J segments on the light chain transgeneare appropriate recombination signal sequences (RSS's). In addition,such transgenes also include appropriate RNA splicing signals to join aconstant region gene segment with the VJ or VDJ rearranged variableregion.

In order to facilitate isotype switching within a heavy chain transgenecontaining more than one C region gene segment, e.g. Cμ and Cγ1 from thehuman genome, as explained below “switch regions” are incorporatedupstream from each of the constant region gene segments and downstreamfrom the variable region gene segments to permit recombination betweensuch constant regions to allow for immunoglobulin class switching, e.g.from IgM to IgG. Such heavy and light immunoglobulin transgenes alsocontain transcription control sequences including promoter regionssituated upstream from the variable region gene segments which typicallycontain TATA motifs. A promoter region can be defined approximately as aDNA sequence that, when operably linked to a downstream sequence, canproduce transcription of the downstream sequence. Promoters may requirethe presence of additional linked cis-acting sequences in order toproduce efficient transcription. In addition, other sequences thatparticipate in the transcription of sterile transcripts are preferablyincluded. Examples of sequences that participate in expression ofsterile transcripts can be found in the published literature, includingRothman et al., Intl. Immunol. 2:621-627 (1990); Reid et al., Proc.Natl. Acad. Sci. USA 86:840-844 (1989); Stavnezer et al., Proc. Natl.Acad. Sci. USA 85:7704-7708 (1988); and Mills et al., Nucl. Acids Res.18:7305-7316 (1991), each of which is incorporated herein by reference.These sequences typically include about at least 50 bp immediatelyupstream of a switch region, preferably about at least 200 bp upstreamof a switch region; and more preferably about at least 200-1000 bp ormore upstream of a switch region. Suitable sequences occur immediatelyupstream of the human S_(γ1), S_(γ2), S_(γ3), S_(γ4), S_(α1), S_(α2),and S_(ε) switch regions; the sequences immediately upstream of thehuman S_(γ1), and S_(γ3) switch regions can be used to advantage, withS_(γ1) generally preferred. Alternatively, or in combination, murine Igswitch sequences may be used; it may frequently be advantageous toemploy Ig switch sequences of the same species as the transgenicnon-human animal. Furthermore, interferon (IFN) inducibletranscriptional regulatory elements, such as IFN-inducible enhancers,are preferably included immediately upstream of transgene switchsequences.

In addition to promoters, other regulatory sequences which functionprimarily in B-lineage cells are used. Thus, for example, a light chainenhancer sequence situated preferably between the J and constant regiongene segments on the light chain transgene is used to enhance transgeneexpression, thereby facilitating allelic exclusion. In the case of theheavy chain transgene, regulatory enhancers and also employed. Suchregulatory sequences are used to maximize the transcription andtranslation of the transgene so as to induce allelic exclusion and toprovide relatively high levels of transgene expression.

Although the foregoing promoter and enhancer regulatory controlsequences have been generically described, such regulatory sequences maybe heterologous to the nonhuman animal being derived from the genomicDNA from which the heterologous transgene immunoglobulin gene segmentsare obtained. Alternately, such regulatory gene segments are derivedfrom the corresponding regulatory sequences in the genome of thenon-human animal, or closely related species, which contains the heavyand light transgene.

In the preferred embodiments, gene segments are derived from humanbeings. The transgenic non-human animals harboring such heavy and lighttransgenes are capable of mounting an Ig-mediated immune response to aspecific antigen administered to such an animal. B-cells are producedwithin such an animal which are capable of producing heterologous humanantibody. After immortalization, and the selection for an appropriatemonoclonal antibody (Mab), e.g. a hybridoma, a source of therapeutichuman monoclonal antibody is provided. Such human Mabs havesignificantly reduced immunogenicity when therapeutically administeredto humans.

Although the preferred embodiments disclose the construction of heavyand light transgenes containing human gene segments, the invention isnot so limited. In this regard, it is to be understood that theteachings described herein may be readily adapted to utilizeimmunoglobulin gene segments from a species other than human beings. Forexample, in addition to the therapeutic treatment of humans with theantibodies of the invention, therapeutic antibodies encoded byappropriate gene segments may be utilized to generate monoclonalantibodies for use in the veterinary sciences.

Rearranged Transgenes

In an alternative embodiment, transgenic nonhuman animals containfunctionally at least one rearranged heterologous heavy chainimmunoglobulin transgene in the germline of the transgenic animal. Suchanimals contain primary repertoire B-cells that express such rearrangedheavy transgenes. Such B-cells preferably are capable of undergoingsomatic mutation when contacted with an antigen to form a heterologousantibody having high affinity and specificity for the antigen. Saidrearranged transgenes will contain at least two C_(H) genes and theassociated sequences required for isotype switching.

The invention also includes transgenic animals containing germ linecells having heavy and light transgenes wherein one of the saidtransgenes contains rearranged gene segments with the other containingunrearranged gene segments. In such animals, the heavy chain transgenesshall have at least two C_(H) genes and the associated sequencesrequired for isotype switching.

The invention further includes methods for generating a syntheticvariable region gene segment repertoire to be used in the transgenes ofthe invention. The method comprises generating a population ofimmunoglobulin V segment DNAs wherein each of the V segment DNAs encodesan immunoglobulin V segment and contains at each end a cleavagerecognition site of a restriction endonuclease. The population ofimmunoglobulin V segment DNAs is thereafter concatenated to form thesynthetic immunoglobulin V segment repertoire. Such synthetic variableregion heavy chain transgenes shall have at least two C_(H) genes andthe associated sequences required for isotype switching.

Isotype Switching

In the development of a B lymphocyte, the cell initially produces IgMwith a binding specificity determined by the productively rearrangedV_(H) and V_(L) regions. Subsequently, each B cell and its progeny cellssynthesize antibodies with the same L and H chain V regions, but theymay switch the isotype of the H chain.

The use of μ or δ constant regions is largely determined by alternatesplicing, permitting IgM and IgD to be coexpressed in a single cell. Theother heavy chain isotypes (γ, α, and ε) are only expressed nativelyafter a gene rearrangement event deletes the Cμ and Cδ exons. This generearrangement process, termed isotype switching, typically occurs byrecombination between so called switch segments located immediatelyupstream of each heavy chain gene (except δ). The individual switchsegments are between 2 and 10 kb in length, and consist primarily ofshort repeated sequences. The exact point of recombination differs forindividual class switching events. Investigations which have usedsolution hybridization kinetics or Southern blotting with cDNA-derivedC_(H) probes have confirmed that switching can be associated with lossof C_(H) sequences from the cell.

The switch (S) region of the μ gene, S_(μ), is located about 1 to 2 kb5′ to the coding sequence and is composed of numerous tandem repeats ofsequences of the form (GAGCT)_(n)(GGGGT) (SEQ ID NO: 9-24), where n isusually 2 to 5 but can range as high as 17. (See T. Nikaido et al.Nature 292:845-848 (1981))

Similar internally repetitive switch sequences spanning severalkilobases have been found 5′ of the other C_(H) genes. The S_(α) regionhas been sequenced and found to consist of tandemly repeated 80-bphomology units, whereas murine S_(γ2) a, S_(γ2) b, and S_(γ3) allcontain repeated 49-bp homology units very similar to each other. (See,P. Szurek et al., J. Immunol 135:620-626 (1985) and T. Nikaido et al.,J. Biol. Chem. 257:7322-7329 (1982), which are incorporated herein byreference.) All the sequenced S regions include numerous occurrences ofthe pentamers GAGCT and GGGGT that are the basic repeated elements ofthe S_(μ) gene (T. Nikaido et al., J. Biol. Chem. 257:7322-7329 (1982)which is incorporated herein by reference); in the other S regions thesepentamers are not precisely tandemly repeated as in S_(μ), but insteadare embedded in larger repeat units. The S_(γ1) region has an additionalhigher-order structure: two direct repeat sequences flank each of twoclusters of 49-bp tandem repeats. (See M. R. Mowatt et al., J. Immunol.136:2674-2683 (1986), which is incorporated herein by reference).

Switch regions of human H chain genes have been found to be very similarto their mouse homologs. Indeed, similarity between pairs of human andmouse clones 5′ to the C_(H) genes has been found to be confined to theS regions, a fact that confirms the biological significance of theseregions.

A switch recombination between μ and α genes produces a compositeS_(μ)-S_(α) sequence. Typically, there is no specific site, either inS_(μ) or in any other S region, where the recombination always occurs.

Generally, unlike the enzymatic machinery of V-J recombination, theswitch machinery can apparently accommodate different alignments of therepeated homologous regions of germline S precursors and then join thesequences at different positions within the alignment. (See, T. H.Rabbits et al., Nucleic Acids Res. 9:4509-4524 (1981) and J. Ravetch etal., Proc. Natl. Acad. Sci. USA 77:6734-6738 (1980), which areincorporated herein by reference.)

The exact details of the mechanism(s) of selective activation ofswitching to a particular isotype are unknown. Although exogenousinfluences such as lymphokines and cytokines might upregulateisotype-specific recombinases, it is also possible that the sameenzymatic machinery catalyzes switches to all isotypes and thatspecificity lies in targeting this machinery to specific switch regions.

The T-cell-derived lymphokines IL-4 and IFN_(γ) have been shown tospecifically promote the expression of certain isotypes: in the mouse,IL-4 decreases IgM, IgG2a, IgG2b, and IgG3 expression and increases IgEand IgG1 expression; while IFN_(γ) selectively stimulates IgG2aexpression and antagonizes the IL-4-induced increase in IgE and IgG1expression (Coffman et al., J. Immunol. 136: 949 (1986) and Snapper etal., Science 236: 944 (1987), which are incorporated herein byreference). A combination of IL-4 and IL-5 promotes IgA expression(Coffman et al., J. Immunol. 139: 3685 (1987), which is incorporatedherein by reference).

Most of the experiments implicating T-cell effects on switching have notruled out the possibility that the observed increase in cells withparticular switch recombinations might reflect selection of preswitchedor precommitted cells; but the most likely explanation is that thelymphokines actually promote switch recombination.

Induction of class switching appears to be associated with steriletranscripts that initiate upstream of the switch segments (Lutzker etal., Mol. Cell. Biol. 8:1849 (1988); Stavnezer et al., Proc. Natl. Acad.Sci. USA 85:7704 (1988); Esser and Radbruch, EMBO J. 8:483 (1989);Berton et al., Proc. Natl. Acad. Sci. USA 86:2829 (1989); Rothman etal., Int. Immunol. 2:621 (1990), each of which is incorporated byreference). For example, the observed induction of the γ1 steriletranscript by IL-4 and inhibition by IFN-γ correlates with theobservation that IL-4 promotes class switching to γ1 in B-cells inculture, while IFN-γ inhibits γ1 expression. Therefore, the inclusion ofregulatory sequences that affect the transcription of steriletranscripts may also affect the rate of isotype switching. For example,increasing the transcription of a particular sterile transcripttypically can be expected to enhance the frequency of isotype switchrecombination involving adjacent switch sequences.

For these reasons, it is preferable that transgenes incorporatetranscriptional regulatory sequences within about 1-2 kb upstream ofeach switch region that is to be utilized for isotype switching. Thesetranscriptional regulatory sequences preferably include a promoter andan enhancer element, and more preferably include the 5′ flanking (i.e.,upstream) region that is naturally associated (i.e., occurs in germlineconfiguration) with a switch region. This 5′ flanking region istypically about at least 50 nucleotides in length, preferably about atleast 200 nucleotides in length, and more preferably at least 500-1000nucleotides.

Although a 5′ flanking sequence from one switch region can be operablylinked to a different switch region for transgene construction (e.g., a5′ flanking sequence from the human S_(γ1) switch can be graftedimmediately upstream of the S_(α1) switch; a murine S_(γ1) flankingregion can be grafted adjacent to a human γ1 switch sequence; or themurine S_(γ1) switch can be grafted onto the human γ1 coding region), insome embodiments it is preferred that each switch region incorporated inthe transgene construct have the 5′ flanking region that occursimmediately upstream in the naturally occurring germline configuration.

Monoclonal Antibodies

Monoclonal antibodies can be obtained by various techniques familiar tothose skilled in the art. Briefly, spleen cells from an animal immunizedwith a desired antigen are immortalized, commonly by fusion with amyeloma cell (see, Kohler and Milstein, Eur. J. Immunol., 6:511-519(1976)). Alternative methods of immortalization include transformationwith Epstein Barr Virus, oncogenes, or retroviruses, or other methodswell known in the art. Colonies arising from single immortalized cellsare screened for production of antibodies of the desired specificity andaffinity for the antigen, and yield of the monoclonal antibodiesproduced by such cells may be enhanced by various techniques, includinginjection into the peritoneal cavity of a vertebrate host. Varioustechniques useful in these arts are discussed, for example, in Harlowand Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.(1988) including: immunization of animals to produce immunoglobulins;production of monoclonal antibodies; labeling immunoglobulins for use asprobes; immunoaffinity purification; and immunoassays.

The Transgenic Primary Repertoire

A. The Human Immunoglobulin Loci

An important requirement for transgene function is the generation of aprimary antibody repertoire that is diverse enough to trigger asecondary immune response for a wide range of antigens. The rearrangedheavy chain gene consists of a signal peptide exon, a variable regionexon and a tandem array of multi-domain constant region regions, each ofwhich is encoded by several exons. Each of the constant region genesencode the constant portion of a different class of immunoglobulins.During B-cell development, V region proximal constant regions aredeleted leading to the expression of new heavy chain classes. For eachheavy chain class, alternative patterns of RNA splicing give rise toboth transmembrane and secreted immunoglobulins.

The human heavy chain locus is estimated to consist of approximately 200V gene segments (current data supports the existence of about 50-100 Vgene segments) spanning 2 Mb, approximately 30 D gene segments spanningabout 40 kb, six J segments clustered within a 3 kb span, and nineconstant region gene segments spread out over approximately 300 kb. Theentire locus spans approximately 2.5 Mb of the distal portion of thelong arm of chromosome 14.

B. Gene Fragment Transgenes

1. Heavy Chain Transgene

In a preferred embodiment, immunoglobulin heavy and light chaintransgenes comprise unrearranged genomic DNA from humans. In the case ofthe heavy chain, a preferred transgene comprises a NotI fragment havinga length between 670 to 830 kb. The length of this fragment is ambiguousbecause the 3′ restriction site has not been accurately mapped. It isknown, however, to reside between the α1 and .phi.α gene segments. Thisfragment contains members of all six of the known V_(H) families, the Dand J gene segments, as well as the μ, δ, γ3, γ1 and α1 constant regions(Berman et al., EMBO J. 7:727-738 (1988), which is incorporated hereinby reference). A transgenic mouse line containing this transgenecorrectly expresses a heavy chain class required for B-cell development(IgM) and at least one switched heavy chain class (IgG₁), in conjunctionwith a sufficiently large repertoire of variable regions to trigger asecondary response for most antigens.

2. Light Chain Transgene

A genomic fragment containing all of the necessary gene segments andregulatory sequences from a human light chain locus may be similarlyconstructed. Such transgenes are constructed as described in theExamples and in copending application, entitled “Transgenic Non-HumanAnimals Capable of Producing Heterologous Antibodies,” filed Aug. 29,1990, under U.S. Ser. No. 07/574,748.

C. Transgenes Generated Intracellularly by In Vivo Recombination

It is not necessary to isolate the all or part of the heavy chain locuson a single DNA fragment. Thus, for example, the 670-830 kb NotIfragment from the human immunoglobulin heavy chain locus may be formedin vivo in the non-human animal during transgenesis. Such in vivotransgene construction is produced by introducing two or moreoverlapping DNA fragments into an embryonic nucleus of the non-humananimal. The overlapping portions of the DNA fragments have DNA sequenceswhich are substantially homologous. Upon exposure to the recombinasescontained within the embryonic nucleus, the overlapping DNA fragmentshomologously recombined in proper orientation to form the 670-830 kbNotI heavy chain fragment.

In vivo transgene construction can be used to form any number ofimmunoglobulin transgenes which because of their size are otherwisedifficult, or impossible, to make or manipulate by present technology.Thus, in vivo transgene construction is useful to generateimmunoglobulin transgenes which are larger than DNA fragments which maybe manipulated by YAC vectors (Murray and Szostak, Nature 305:189-193(1983)). Such in vivo transgene construction may be used to introduceinto a non-human animal substantially the entire immunoglobulin locifrom a species not consisting of the transgenic non-human animal.

In addition to forming genomic immunoglobulin transgenes, in vivohomologous recombination may also be utilized to form “mini-locus”transgenes as described in the Examples.

In the preferred embodiments utilizing in vivo transgene construction,each overlapping DNA fragment preferably has an overlappingsubstantially homologous DNA sequence between the end portion of one DNAfragment and the end portion of a second DNA fragment. Such overlappingportions of the DNA fragments preferably comprise about 500 bp to about2000 bp, most preferably 1.0 kb to 2.0 kb. Homologous recombination ofoverlapping DNA fragments to form transgenes in vivo is furtherdescribed in commonly assigned U.S. patent application entitled“Intracellular Generation of DNA by Homologous Recombination of DNAFragments” filed Aug. 29, 1990, under U.S. Ser. No. 07/574,747.

D. Minilocus Transgenes

As used herein, the term “immunoglobulin minilocus” refers to a DNAsequence (which may be within a longer sequence), usually of less thanabout 150 kb, typically between about 25 and 100 kb, containing at leastone each of the following: a functional variable (V) gene segment, afunctional joining (J) region segment, at least one functional constant(C) region gene segment, and—if it is a heavy chain minilocus—afunctional diversity (D) region segment, such that said DNA sequencecontains at least one substantial discontinuity (e.g., a deletion,usually of at least about 2 to 5 kb, preferably 10-25 kb or more,relative to the homologous genomic DNA sequence). A light chainminilocus transgene will be at least 25 kb in length, typically 50 to 60kb. A heavy chain transgene will typically be about 70 to 80 kb inlength, preferably at least about 60 kb with two constant regionsoperably linked to switch regions. Furthermore, the individual elementsof the minilocus are preferably in the germline configuration andcapable of undergoing gene rearrangement in the pre-B cell of atransgenic animal so as to express functional antibody molecules withdiverse antigen specificities encoded entirely by the elements of theminilocus. Further, a heavy chain minilocus comprising at least twoC_(H) genes and the requisite switching sequences is typically capableof undergoing isotype switching, so that functional antibody moleculesof different immunoglobulin classes will be generated. Such isotypeswitching may occur in vivo in B-cells residing within the transgenicnonhuman animal, or may occur in cultured cells of the B-cell lineagewhich have been explanted from the transgenic nonhuman animal.

In an alternate preferred embodiment, immunoglobulin heavy chaintransgenes comprise one or more of each of the V_(H), D, and J_(H) genesegments and two or more of the C_(H) genes. At least one of eachappropriate type gene segment is incorporated into the minilocustransgene. With regard to the C_(H) segments for the heavy chaintransgene, it is preferred that the transgene contain at least one μgene segment and at least one other constant region gene segment, morepreferably a γ gene segment, and most preferably γ3 or γ1. Thispreference is to allow for class switching between IgM and IgG forms ofthe encoded immunoglobulin and the production of a secretable form ofhigh affinity non-IgM immunoglobulin. Other constant region genesegments may also be used such as those which encode for the productionof IgD, IgA and IgE.

Those skilled in the art will also construct transgenes wherein theorder of occurrence of heavy chain C_(H) genes will be different fromthe naturally-occurring spatial order found in the germline of thespecies serving as the donor of the C_(H) genes.

Additionally, those skilled in the art can select C_(H) genes from morethan one individual of a species (e.g., allogeneic C_(H) genes) andincorporate said genes in the transgene as supernumerary C_(H) genescapable of undergoing isotype switching; the resultant transgenicnonhuman animal may then, in some embodiments, make antibodies ofvarious classes including all of the allotypes represented in thespecies from which the transgene C_(H) genes were obtained.

Still further, those skilled in the art can select C_(H) genes fromdifferent species to incorporate into the transgene. Functional switchsequences are included with each C_(H) gene, although the switchsequences used are not necessarily those which occur naturally adjacentto the C_(H) gene. Interspecies C_(H) gene combinations will produce atransgenic nonhuman animal which may produce antibodies of variousclasses corresponding to C_(H) genes from various species. Transgenicnonhuman animals containing interspecies C_(H) transgenes may serve asthe source of B-cells for constructing hybridomas to produce monoclonalsfor veterinary uses.

The heavy chain J region segments in the human comprise six functional Jsegments and three pseudo genes clustered in a 3 kb stretch of DNA.Given its relatively compact size and the ability to isolate thesesegments together with the μ gene and the 5′ portion of the δ gene on asingle 23 kb SFiI/SpeI fragment (Sado et al., Biochem. Biophys. Res.Comm. 154:264271 (1988), which is incorporated herein by reference), itis preferred that all of the J region gene segments be used in themini-locus construct. Since this fragment spans the region between the μand δ genes, it is likely to contain all of the 3′ cis-linked regulatoryelements required for μ expression. Furthermore, because this fragmentincludes the entire J region, it contains the heavy chain enhancer andthe μ switch region (Mills et al., Nature 306:809 (1983); Yancopoulosand Alt, Ann. Rev. Immunol. 4:339-368 (1986), which are incorporatedherein by reference). It also contains the transcription start siteswhich trigger VDJ joining to form primary repertoire B-cells(Yancopoulos and Alt, Cell 40:271-281 (1985), which is incorporatedherein by reference). Alternatively, a 36 kb BssHII/SpeIl fragment,which includes part on the D region, may be used in place of the 23 kbSfiI/SpeIl fragment. The use of such a fragment increases the amount of5′ flanking sequence to facilitate efficient D-to-J joining.

The human D region consists of 4 homologous 9 kb subregions, linked intandem (Siebenlist, et al. (1981), Nature, 294, 631-635). Each subregioncontains up to 10 individual D segments. Some of these segments havebeen mapped and are shown in FIG. 4. Two different strategies are usedto generate a mini-locus D region. The first strategy involves usingonly those D segments located in a short contiguous stretch of DNA thatincludes one or two of the repeated D subregions. A candidate is asingle 15 kb fragment that contains 12 individual D segments. This pieceof DNA consists of 2 contiguous EcoRI fragments and has been completelysequenced (Ichihara, et al. (1988), EMBO J., 7, 4141-4150). Twelve Dsegments should be sufficient for a primary repertoire. However, giventhe dispersed nature of the D region, an alternative strategy is toligate together several non-contiguous D-segment containing fragments,to produce a smaller piece of DNA with a greater number of segments.Additional D-segment genes can be identified, for example, by thepresence of characteristic flanking nonamer and heptamer sequences,supra, and by reference to the literature.

At least one, and preferably more than one V gene segment is used toconstruct the heavy chain minilocus transgene. Rearranged orunrearranged V segments with or without flanking sequences can beisolated as described in copending applications, U.S. Ser. No.07/574,748 filed Aug. 29, 1990, PCT/US91/06185 filed Aug. 28, 1991, andU.S. Ser. No. 07/810,279 filed Dec. 17, 1991, each of which isincorporated herein by reference.

Rearranged or unrearranged V segments, D segments, J segments, and Cgenes, with or without flanking sequences, can be isolated as describedin copending applications U.S. Ser. No. 07/574,748 filed Aug. 29, 1990and PCT/US91/06185 filed Aug. 28, 1991.

A minilocus light chain transgene may be similarly constructed from thehuman λ or κ immunoglobulin locus. Thus, for example, an immunoglobulinheavy chain minilocus transgene construct, e.g., of about 75 kb,encoding V, D, J and constant region sequences can be formed from aplurality of DNA fragments, with each sequence being substantiallyhomologous to human gene sequences. Preferably, the sequences areoperably linked to transcription regulatory sequences and are capable ofundergoing rearrangement. With two or more appropriately placed constantregion sequences (e.g., μ and γ) and switch regions, switchrecombination also occurs. An exemplary light chain transgene constructcan be formed similarly from a plurality of DNA fragments, substantiallyhomologous to human DNA and capable of undergoing rearrangement, asdescribed in copending application, U.S. Ser. No. 07/574,748 filed Aug.29, 1990.

E. Transgene Constructs Capable of Isotype Switching

Ideally, transgene constructs that are intended to undergo classswitching should include all of the cis-acting sequences necessary toregulate sterile transcripts. Naturally occurring switch regions andupstream promoters and regulatory sequences (e.g., IFN-inducibleelements) are preferred cis-acting sequences that are included intransgene constructs capable of isotype switching. About at least 50basepairs, preferably about at least 200 basepairs, and more preferablyat least 500 to 1000 basepairs or more of sequence immediately upstreamof a switch region, preferably a human γ1 switch region, should beoperably linked to a switch sequence, preferably a human γ1 switchsequence. Further, switch regions can be linked upstream of (andadjacent to) C_(H) genes that do not naturally occur next to theparticular switch region. For example, but not for limitation, a humanγ1 switch region may be linked upstream from a human α₂ C_(H) gene, or amurine γ₁ switch may be linked to a human C_(H) gene.

An alternative method for obtaining non-classical isotype switching(e.g., δ-associated deletion) in transgenic mice involves the inclusionof the 400 bp direct repeat sequences (Σμ and εμ) that flank the human μgene (Yasui et al., Eur. J. Immunol. 19:1399 (1989)). Homologousrecombination between these two sequences deletes the μ gene in IgD-onlyB-cells. Heavy chain transgenes can be represented by the followingformulaic description:(V_(H))_(x)-(D)_(y)-(J_(H))_(z)-(S_(D))_(m)-(C₁)_(n)-([(T)-(S_(A))_(p)-(C₂)]_(q)where:

V_(H) is a heavy chain variable region gene segment,

D is a heavy chain D (diversity) region gene segment,

J_(H) is a heavy chain J (joining) region gene segment,

S_(D) is a donor region segment capable of participating in arecombination event with the S_(a) acceptor region segments such thatisotype switching occurs,

C₁ is a heavy chain constant region gene segment encoding an isotypeutilized in for B cell development (e.g., μ or δ),

T is a cis-acting transcriptional regulatory region segment containingat least a promoter,

S_(A) is an acceptor region segment capable of participating in arecombination event with selected S_(D) donor region segments, such thatisotype switching occurs,

C₂ is a heavy chain constant region gene segment encoding an isotypeother than μ (e.g., γ₁, γ₂, γ₃, γ₄, α₁, α₂, ε).

x, y, z, m, n, p, and q are integers. x is 1-100, n is 0-10, y is 1-50,p is 1-10, z is 1-50, q is 0-50, m is 0-10. Typically, when thetransgene is capable of isotype switching, q must be at least 1, m is atleast 1, n is at least 1, and m is greater than or equal to n.

V_(H), D, J_(H), S_(D), C₁, T, S_(A), and C_(Z) segments may be selectedfrom various species, preferably mammalian species, and more preferablyfrom human and murine germline DNA.

V_(H) segments may be selected from various species, but are preferablyselected from V_(H) segments that occur naturally in the human germline,such as V_(H) 251. Typically about 2 V_(H) gene segments are included,preferably about 4 V_(H) segments are included, and most preferably atleast about 10 V_(H) segments are included.

At least one D segment is typically included, although at least 10 Dsegments are preferably included, and some embodiments include more thanten D segments. Some preferred embodiments include human D segments.

Typically at least one J_(H) segment is incorporated in the transgene,although it is preferable to include about six J_(H) segments, and somepreferred embodiments include more than about six J_(H) segments. Somepreferred embodiments include human J_(H) segments, and furtherpreferred embodiments include six human J_(H) segments and no nonhumanJ_(H) segments.

S_(D) segments are donor regions capable of participating inrecombination events with the S_(A) segment of the transgene. Forclassical isotype switching, S_(D) and S_(A) are switch regions such asS_(μ), S_(γ1), S_(γ2), S_(γ3), S_(γ4), S_(α), S_(α2), and S_(ε).Preferably the switch regions are murine or human, more preferably S_(D)is a human or murine S_(μ) and S_(A) is a human or murine S_(γ1). Fornonclassical isotype switching (δ-associated deletion), S_(D) and S_(A)are preferably the 400 basepair direct repeat sequences that flank thehuman μ gene.

C₁ segments are typically μ or δ genes, preferably a μ gene, and morepreferably a human or murine μ gene.

T segments typically include S′ flanking sequences that are adjacent tonaturally occurring (i.e., germline) switch regions. T segmentstypically at least about at least 50 nucleotides in length, preferablyabout at least 200 nucleotides in length, and more preferably at least500-1000 nucleotides in length. Preferably T segments are 5′ flankingsequences that occur immediately upstream of human or murine switchregions in a germline configuration. It is also evident to those ofskill in the art that T segments may comprise cis-acting transcriptionalregulatory sequences that do not occur naturally in an animal germline(e.g., viral enhancers and promoters such as those found in SV40,adenovirus, and other viruses that infect eukaryotic cells).

C₂ segments are typically a γ₁, γ₂, γ₃, γ₄, α₁, α₂, or ε C_(H) gene,preferably a human c^(H) gene of these isotypes, and more preferably ahuman γ₁ or γ₃ gene. Murine γ_(2a) and γ_(2b) may also be used, as maydownstream (i.e., switched) isotype genes form various species. Wherethe heavy chain transgene contains an immunoglobulin heavy chainminilocus, the total length of the transgene will be typically 150 kilobasepairs or less.

In general, the transgene will be other than a native heavy chain Iglocus. Thus, for example, deletion of unnecessary regions orsubstitutions with corresponding regions from other species will bepresent.

F. Methods for Determining Functional Isotype Switching in Ig Transgenes

The occurrence of isotype switching in a transgenic nonhuman animal maybe identified by any method known to those * in the art. Preferredembodiments include the following, employed either singly or incombination:

1. detection of mRNA transcripts that contain a sequence homologous toat least one transgene downstream C_(H) gene other than δ and anadjacent sequence homologous to a transgene V_(H)-D_(H)-J_(H) rearrangedgene; such detection may be by Northern hybridization, S₁ nucleaseprotection assays, PCR amplification, cDNA cloning, or other methods;

2. detection in the serum of the transgenic animal, or in supernatantsof cultures of hybridoma cells made from B-cells of the transgenicanimal, of immunoglobulin proteins encoded by downstream C_(H) genes,where such proteins can also be shown by immunochemical methods tocomprise a functional variable region;

3. detection, in DNA from B-cells of the transgenic animal or in genomicDNA from hybridoma cells, of DNA rearrangements consistent with theoccurrence of isotype switching in the transgene, such detection may beaccomplished by Southern blot hybridization, PCR amplification, genomiccloning, or other method; or

4. identification of other indicia of isotype switching, such asproduction of sterile transcripts, production of characteristic enzymesinvolved in switching (e.g., “switch recombinase”), or othermanifestations that may be detected, measured, or observed bycontemporary techniques.

Because each transgenic line may represent a different site ofintegration of the transgene, and a potentially different tandem arrayof transgene inserts, and because each different configuration oftransgene and flanking DNA sequences can affect gene expression, it ispreferable to identify and use lines of mice that express high levels ofhuman immunoglobulins, particularly of the IgG isotype, and contain theleast number of copies of the transgene. Single copy transgenicsminimize the potential problem of incomplete allelic expression.Transgenes are typically integrated into host chromosomal DNA, mostusually into germline DNA and propagated by subsequent breeding ofgermline transgenic breeding stock animals. However, other vectors andtransgenic methods known in the present art or subsequently developedmay be substituted as appropriate and as desired by a practitioner.

Trans-switching to endogenous nonhuman heavy chain constant region genescan occur and produce chimeric heavy chains and antibodies comprisingsuch chimeric human/mouse heavy chains. Such chimeric antibodies may bedesired for certain uses described herein or may be undesirable.

G. Functional Disruption of Endogenous Immunoglobulin Loci

The expression of successfully rearranged immunoglobulin heavy and lighttransgenes is expected to have a dominant effect by suppressing therearrangement of the endogenous immunoglobulin genes in the transgenicnonhuman animal. However, another way to generate a nonhuman that isdevoid of endogenous antibodies is by mutating the endogenousimmunoglobulin loci. Using embryonic stem cell technology and homologousrecombination, the endogenous immunoglobulin repertoire can be readilyeliminated. The following describes the functional description of themouse immunoglobulin loci. The vectors and methods disclosed, however,can be readily adapted for use in other non-human animals.

Briefly, this technology involves the inactivation of a gene, byhomologous recombination, in a pluripotent cell line that is capable ofdifferentiating into germ cell tissue. A DNA construct that contains analtered, copy of a mouse immunoglobulin gene is introduced into thenuclei of embryonic stem cells. In a portion of the cells, theintroduced DNA recombines with the endogenous copy of the mouse gene,replacing it with the altered copy. Cells containing the newlyengineered genetic lesion are injected into a host mouse embryo, whichis reimplanted into a recipient female. Some of these embryos developinto chimeric mice that possess germ cells entirely derived from themutant cell line. Therefore, by breeding the chimeric mice it ispossible to obtain a new line of mice containing the introduced geneticlesion (reviewed by Capecchi (1989), Science, 244, 1288-1292).

Because the mouse λ locus contributes to only 5% of the immunoglobulins,inactivation of the heavy chain and/or κ-light chain loci is sufficient.There are three ways to disrupt each of these loci, deletion of the Jregion, deletion of the J-C intron enhancer, and disruption of constantregion coding sequences by the introduction of a stop codon. The lastoption is the most straightforward, in terms of DNA construct design.Elimination of the μ gene disrupts B-cell maturation thereby preventingclass switching to any of the functional heavy chain segments. Thestrategy for knocking out these loci is outlined below.

To disrupt the mouse μ and κ genes, targeting vectors are used based onthe design employed by Jaenisch and co-workers (Zijlstra, et al. (1989),Nature, 342, 435-438) for the successful disruption of the mouseβ2-microglobulin gene. The neomycin resistance gene (neo), from theplasmid pMCIneo is inserted into the coding region of the target gene.The pMCIneo insert uses a hybrid viral promoter/enhancer sequence todrive neo expression. This promoter is active in embryonic stem cells.Therefore, neo can be used as a selectable marker for integration of theknock-out construct. The HSV thymidine kinase (tk) gene is added to theend of the construct as a negative selection marker against randominsertion events (Zijlstra, et al., supra.).

A preferred strategy for disrupting the heavy chain locus is theelimination of the J region. This region is fairly compact in the mouse,spanning only 1.3 kb. To construct a gene targeting vector, a 15 kb KpnIfragment containing all of the secreted A constant region exons frommouse genomic library is isolated. The 1.3 kb J region is replaced withthe 1.1 kb insert from pMCIneo. The HSV tk gene is then added to the 5′end of the KpnI fragment. Correct integration of this construct, viahomologous recombination, will result in the replacement of the mouseJ_(H) region with the neo gene. Recombinants are screened by PCR, usinga primer based on the neo gene and a primer homologous to mousesequences 5′ of the KpnI site in the D region.

Alternatively, the heavy-chain locus is knocked out by disrupting thecoding region of the μ gene. This approach involves the same 15 kb KpnIfragment used in the previous approach. The 1.1 kb insert from pMCIneois inserted at a unique BamHI site in exon II, and the HSV tk gene addedto the 3′ KpnI end. Double crossover events on either side of the neoinsert, that eliminate the tk gene, are then selected for. These aredetected from pools of selected clones by PCR amplification. One of thePCR primers is derived from neo sequences and the other from mousesequences outside of the targeting vector. The functional disruption ofthe mouse immunoglobulin loci is presented in the Examples.

G. Suppressing Expression of Endogenous Immunoglobulin Loci

In addition to functional disruption of endogenous Ig loci, analternative method for preventing the expression of an endogenous Iglocus is suppression. Suppression of endogenous Ig genes may beaccomplished with antisense RNA produced from one or more integratedtransgenes, by antisense oligonucleotides, and/or by administration ofantisera specific for one or more endogenous Ig chains.

Antisense Polynucleotides

Antisense RNA transgenes can be employed to partially or totallyknock-out expression of specific genes (Pepin et al. (1991) Nature 355:725; Helene., C. and Toulme, J. (1990) Biochimica Biophys. Acta 1049:99; Stout, J. and Caskey, T. (1990) Somat. Cell Mol. Genet. 16: 369;Munir et al. (1990) Somat. Cell Mol. Genet. 16: 383, each of which isincorporated herein by reference).

“Antisense polynucleotides” are polynucleotides that: (1) arecomplementary to all or part of a reference sequence, such as a sequenceof an endogenous Ig C_(H) or C_(L) region, and (2) which specificallyhybridize to a complementary target sequence, such as a chromosomal genelocus or a Ig mRNA. Such complementary antisense polynucleotides mayinclude nucleotide substitutions, additions, deletions, ortranspositions, so long as specific hybridization to the relevant targetsequence is retained as a functional property of the polynucleotide.Complementary antisense polynucleotides include soluble antisense RNA orDNA oligonucleotides which can hybridize specifically to individual mRNAspecies and prevent transcription and/or RNA processing of the mRNAspecies and/or translation of the encoded polypeptide (Ching et al.,Proc. Natl. Acad. Sci. U.S.A. 86:10006-10010 (1989); Broder et al., Ann.Int. Med. 113:604-618 (1990); Loreau et al., FEBS Letters 274:53-56(1990); Holcenberg et al., WO91/11535; U.S. Ser. No. 07/530,165 (“Newhuman CRIPTO gene”); WO91/09865; WO91/04753; WO90/13641; and EP 386563,each of which is incorporated herein by reference). An antisensesequence is a polynucleotide sequence that is complementary to at leastone immunoglobulin gene sequence of at least about 15 contiguousnucleotides in length, typically at least 20 to 30 nucleotides inlength, and preferably more than about 30 nucleotides in length.However, in some embodiments, antisense sequences may havesubstitutions, additions, or deletions as compared to the complementaryimmunoglobulin gene sequence, so long as specific hybridization isretained as a property of the antisense polynucleotide. Generally, anantisense sequence is complementary to an endogenous immunoglobulin genesequence that encodes, or has the potential to encode after DNArearrangement, an immunoglobulin chain. In some cases, sense sequencescorresponding to an immunoglobulin gene sequence may function tosuppress expression, particularly by interfering with transcription.

The antisense polynucleotides therefore inhibit production of theencoded polypeptide(s). In this regard, antisense polynucleotides thatinhibit transcription and/or translation of one or more endogenous Igloci can alter the capacity and/or specificity of a non-human animal toproduce immunoglobulin chains encoded by endogenous Ig loci.

Antisense polynucleotides may be produced from a heterologous expressioncassette in a transfectant cell or transgenic cell, such as a transgenicpluripotent hematopoietic stem cell used to reconstitute all or part ofthe hematopoietic stem cell population of an individual, or a transgenicnonhuman animal. Alternatively, the antisense polynucleotides maycomprise soluble oligonucleotides that are administered to the externalmilieu, either in culture medium in vitro or in the circulatory systemor interstitial fluid in vivo. Soluble antisense polynucleotides presentin the external milieu have been shown to gain access to the cytoplasmand inhibit translation of specific mRNA species. In some embodimentsthe antisense polynucleotides comprise methylphosphonate moieties,alternatively phosphorothiolates or O-methylribonucleotides may be used,and chimeric oligonucleotides may also be used (Dagle et al. (1990)Nucleic Acids Res. 18: 4751). For some applications, antisenseoligonucleotides may comprise polbyamide nucleic acids (Nielsen et al.(1991) Science 254: 1497). For general methods relating to antisensepolynucleotides, see Antisense RNA and DNA, (1988), D. A. Melton, Ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

Antisense polynucleotides complementary to one or more sequences areemployed to inhibit transcription, RNA processing, and/or translation ofthe cognate mRNA species and thereby effect a reduction in the amount ofthe respective encoded polypeptide. Such antisense polynucleotides canprovide a therapeutic function by inhibiting the formation of one ormore endogenous Ig chains in vivo.

Whether as soluble antisense oligonucleotides or as antisense RNAtranscribed from an antisense transgene, the antisense polynucleotidesof this invention are selected so as to hybridize preferentially toendogenous Ig sequences at physiological conditions in vivo. Mosttypically, the selected antisense polynucleotides will not appreciablyhybridize to heterologous Ig sequences encoded by a heavy or light chaintransgene of the invention (i.e., the antisense oligonucleotides willnot inhibit transgene Ig expression by more than about 25 to 35percent).

Antiserum Suppression

Partial or complete suppression of endogenous Ig chain expression can beproduced by injecting mice with antisera against one or more endogenousIg chains (Weiss et al. (1984) Proc. Natl. Acad. Sci. (U.S.A.) 81 211,which is incorporated herein by reference). Antisera are selected so asto react specifically with one or more endogenous (e.g., murine) Igchains but to have minimal or no cross-reactivity with heterologous Igchains encoded by an Ig transgene of the invention. Thus, administrationof selected antisera according to a schedule as typified by that ofWeiss et al. op.cit. will suppress endogenous Ig chain expression butpermits expression of heterologous Ig chain(s) encoded by a transgene ofthe present invention. Suitable antibody sources for antibody comprise:

(1) monoclonal antibodies, such as a monoclonal antibody thatspecifically binds to a murine μ, γ, κ, or λ chains but does not reactwith the human immunoglobulin chain(s) encoded by a human Ig transgeneof the invention;

(2) mixtures of such monoclonal antibodies, so that the mixture bindswith multiple epitopes on a single species of endogenous Ig chain, withmultiple endogenous Ig chains (e.g., murine μ and murine γ, or withmultiple epitopes and multiple chains or endogenous immunoglobulins;

(3) polyclonal antiserum or mixtures thereof, typically suchantiserum/antisera is monospecific for binding to a single species ofendogenous Ig chain (e.g., murine μ, murine γ, murine κ, murine λ) or tomultiple species of endogenous Ig chain, and most preferably suchantisera possesses negligible binding to human immunoglobulin chainsencoded by a transgene of the invention; and/or

(4) a mixture of polyclonal antiserum and monoclonal antibodies bindingto a single or multiple species of endogenous Ig chain, and mostpreferably possessing negligible binding to human immunoglobulin chainsencoded by a transgene of the invention. Generally, polyclonalantibodies are preferred, and such substantially monospecific polyclonalantibodies can be advantageously produced from an antiserum raisedagainst human immunoglobulin(s) by pre-adsorption with antibodiesderived from the nonhuman animal species (e.g., murine) and/or, forexample, by affinity chromatography of the antiserum or purifiedfraction thereof on an affinity resin containing immobilized human Ig(wherein the bound fraction is enriched for the desired anti-human Ig inthe antiserum; the bound fraction is typically eluted with conditions oflow pH or a chaotropic salt solution).

Cell separation and/or complement fixation can be employed to providethe enhancement of antibody-directed cell depletion of lymphocytesexpressing endogenous (e.g., murine) immunoglobulin chains. In oneembodiment, for example, antibodies are employed for ex vivo depletionof murine Ig-expressing explanted hematopoietic cells and/or B-lineagelymphocytes obtained from a transgenic mouse harboring a human Igtransgene. Thus, hematopoietic cells and/or B-lineage lymphocytes areexplanted from a transgenic nonhuman animal harboring a human Igtransgene (preferably harboring both a human heavy chain transgene and ahuman light chain transgene) and the explanted cells are incubated withan antibody (or antibodies) which (1) binds to an endogenousimmunoglobulin (e.g., murine μ and/or κ) and (2) lacks substantialbinding to human immunoglobulin chains encoded by the transgene(s). Suchantibodies are referred to as “suppression antibodies” for clarity. Theexplanted cell population is selectively depleted of cells which bind tothe suppression antibody(ies); such depletion can be accomplished byvarious methods, such as (1) physical separation to remove suppressionantibody-bound cells from unbound cells (e.g., the suppressionantibodies may be bound to a solid support or magnetic bead toimmobilize and remove cells binding to the suppression antibody), (2)antibody-dependent cell killing of cells bound by the suppressionantibody (e.g., by ADCC, by complement fixation, or by a toxin linked tothe suppression antibody), and (3) clonal anergy induced by thesuppression antibody, and the like.

Frequently, antibodies used for antibody suppression of endogenous Igchain production will be capable of fixing complement. It is frequentlypreferable that such antibodies may be selected so as to react well witha convenient complement source for ex vivo/in vitro depletion, such asrabbit or guinea pig complement. For in vivo depletion, it is generallypreferred that the suppressor antibodies possess effector functions inthe nonhuman transgenic animal species; thus, a suppression antibodycomprising murine effector functions (e.g., ADCC and complementfixation) generally would be preferred for use in transgenic mice.

In one variation, a suppression antibody that specifically binds to apredetermined endogenous immunoglobulin chain is used for ex vivo/invitro depletion of lymphocytes expressing an endogenous immunoglobulin.A cellular explant (e.g., lymphocyte sample) from a transgenic nonhumananimal harboring a human immunoglobulin transgene is contacted with asuppression antibody and cells specifically binding to the suppressionantibody are depleted (e.g., by immobilization, complement fixation, andthe like), thus generating a cell subpopulation depleted in cellsexpressing endogenous (nonhuman) immunoglobulins (e.g., lymphocytesexpressing murine Ig). The resultant depleted lymphocyte population (Tcells, human Ig-positive B-cells, etc.) can be transferred into aimmunocompatible (i.e., MHC-compatible) nonhuman animal of the samespecies and which is substantially incapable of producing endogenousantibody (e.g., SCID mice, RAG-1 or RAG-2 knockout mice). Thereconstituted animal (mouse) can then be immunized with an antigen (orreimmunized with an antigen used to immunize the donor animal from whichthe explant was obtained) to obtain high-affinity (affinity matured)antibodies and B-cells producing such antibodies. Such B-cells may beused to generate hybridomas by conventional cell fusion and screened.Antibody suppression can be used in combination with other endogenous Iginactivation/suppression methods (e.g., J_(H) knockout, C_(H) knockout,D-region ablation, antisense suppression, compensated frameshiftinactivation).

Complete Endogenous Ig Locus Inactivation

In certain embodiments, it is desirable to effect complete inactivationof the endogenous Ig loci so that hybrid immunoglobulin chainscomprising a human variable region and a non-human (e.g., murine)constant region cannot be formed (e.g., by trans-switching between thetransgene and endogenous Ig sequences). Knockout mice bearing endogenousheavy chain alleles with are functionally disrupted in the J_(H) regiononly frequently exhibit trans-switching, typically wherein a rearrangedhuman variable region (VDJ) encoded by a transgene is expressed as afusion protein linked to an endogenous murine constant region, althoughother trans-switched junctions are possible. To overcome this potentialproblem, it is generally desirable to completely inactivate theendogenous heavy chain locus by any of various methods, including butnot limited to the following: (1) functionally disrupting and/ordeleting by homologous recombination at least one and preferably all ofthe endogenous heavy chain constant region genes, (2) mutating at leastone and preferably all of the endogenous heavy chain constant regiongenes to encode a termination codon (or frameshift) to produce atruncated or frameshifted product (if trans-switched), and other methodsand strategies apparent to those of skill in the art. Deletion of asubstantial portion or all of the heavy chain constant region genesand/or D-region genes may be accomplished by various methods, includingsequential deletion by homologous recombination targeting vectors,especially of i the “hit-and-run” type and the like. Similarly,functional disruption and/or deletion of at least one endogenous lightchain locus (e.g., κ) to ablate endogenous light chain constant regiongenes is often preferable.

Frequently, it is desirable to employ a frameshifted transgene whereinthe heterologous transgene comprises a frameshift in the J segment(s)and a compensating frameshift (i.e., to regenerate the original readingframe) in the initial region (i.e., amino-terminal coding portion) ofone or more (preferably all) of the transgene constant region genes.Trans-switching to an endogenous IgH locus constant gene (which does notcomprise a compensating frameshift) will result in a truncated ormissense product that results in the trans-switched B cell being deletedor non-selected, thus suppressing the trans-switched phenotype.

Antisense suppression and antibody suppression may also be used toeffect a substantially complete functional inactivation of endogenous Iggene product expression (e.g., murine heavy and light chain sequences)and/or trans-switched antibodies (e.g., human variable/murine constantchimeric antibodies).

Various combinations of the inactivation and suppression strategies maybe used to effect essentially total suppression of endogenous (e.g.,murine) Ig chain expression.

Trans-Switching

In some variations, it may be desirable to produce a trans-switchedimmunoglobulin. For example, such trans-switched heavy chains can bechimeric (i.e., a non-murine (human) variable region and a murineconstant region). Antibodies comprising such chimeric trans-switchedimmunoglobulins can be used for a variety of applications where it isdesirable to have a non-human (e.g., murine) constant region (e.g., forretention of effector functions in the host, for the presence of murineimmunological determinants such as for binding of a secondary antibodywhich does not bind human constant regions). For one example, a humanvariable region repertoire may possess advantages as compared to themurine variable region repertoire with respect to certain antigens.Presumably the human V_(H), D, J_(H), V_(L), and J_(L) genes have beenselected for during evolution for their ability to encodeimmunoglobulins that bind certain evolutionarily important antigens;antigens which provided evolutionary selective pressure for the murinerepertoire can be distinct from those antigens which providedevolutionary pressure to shape the human repertoire. Other repertoireadvantages may exist, making the human variable region repertoireadvantageous when combined with a murine constant region (e.g., atrans-switched murine) isotype. The presence of a murine constant regioncan afford advantages over a human constant region. For example, amurine γ constant region linked to a human variable region bytrans-switching may provide an antibody which possesses murine effectorfunctions (e.g., ADCC, murine complement fixation) so that such achimeric antibody (preferably monoclonal) which is reactive with apredetermined antigen (e.g., human IL-2 receptor) may be tested in amouse disease model, such as a mouse model of graft-versus-host diseasewherein the T lymphocytes in the mouse express a functional human IL-2receptor. Subsequently, the human variable region encoding sequence maybe isolated (e.g., by PCR amplification or cDNA cloning from the source(hybridoma clone)) and spliced to a sequence encoding a desired humanconstant region to encode a human sequence antibody more suitable forhuman therapeutic uses where immunogenicity is preferably minimized. Thepolynucleotide(s) having the resultant fully human encoding sequence(s)can be expressed in a host cell (e.g., from an expression vector in amammalian cell) and purified for pharmaceutical formulation. For someapplications, the chimeric antibodies may be used directly withoutreplacing the murine constant region with a human constant region. Othervariations and uses of trans-switched chimeric antibodies will beevident to those of skill in the art.

The present invention provides transgenic nonhuman animals containing Blymphocytes which express chimeric antibodies, generally resulting fromtrans-switching between a human heavy chain transgene and an endogenousmurine heavy chain constant region gene. Such chimeric antibodiescomprise a human sequence variable region and a murine constant region,generally a murine switched (i.e., non-μ, non-δ) isotype. The transgenicnonhuman animals capable of making chimeric antibodies to apredetermined antigen are usually also competent to make fully humansequence antibodies if both human heavy chain and human light chaintransgenes encoding human variable and human constant region genes areintegrated. Most typically, the animal is homozygous for a functionallydisrupted heavy chain locus and/or light chain locus but retains one ormore endogenous heavy chain constant region gene(s) capable oftrans-switching (e.g., γ, α, ε) and frequently retains a cis-linkedenhancer. Such a mouse is immunized with a predetermined antigen,usually in combination with an adjuvant, and an immune responsecomprising a detectable amount of chimeric antibodies comprising heavychains composed of human sequence variable regions linked to murineconstant region sequences is produced. Typically, the serum of such animmunized animal can comprise such chimeric antibodies at concentrationsof about at least 1 μg/ml, often about at least 10 μg/ml, frequently atleast 30 μg/ml, and up to 50 to 100 μg/ml or more. The antiserumcontaining antibodies comprising chimeric human variable/mouse constantregion heavy chains typically also comprises antibodies which comprisehuman variable/human constant region (complete human sequence) heavychains. Chimeric trans-switched antibodies usually comprise (1) achimeric heavy chain composed of a human variable region and a murineconstant region (typically a murine gamma) and (2) a humantransgene-encoded light chain (typically kappa) or a murine light chain(typically lambda in a kappa knockout background). Such chimerictrans-switched antibodies generally bind to a predetermined antigen(e.g., the immunogen) with an affinity of about at least 1×10⁷ M⁻¹,preferably with an affinity of about at least 5×10⁷ M⁻¹, more preferablywith an affinity of at least 1×10⁸ M⁻¹ to 1×10⁹ M⁻¹ or more. Frequently,the predetermined antigen is a human protein, such as for example ahuman dell surface antigen (e.g., CD4, CD8, IL-8, IL-2 receptor, EGFreceptor, PDGF receptor), other human biological macromolecule (e.g.,thrombomodulin, protein C, carbohydrate antigen, sialyl Lewis antigen,L-selectin), or nonhuman disease associated macromolecule (e.g.,bacterial LPS, virion capsid protein or envelope glycoprotein) and thelike.

The invention provides transgenic nonhuman animals comprising a genomecomprising: (1) a homozygous functionally disrupted endogenous heavychain locus comprising at least one murine constant region gene capableof trans-switching (e.g., in cis linkage to a functional switchrecombination sequence and typically to a functional enhancer), (2) ahuman heavy chain transgene capable of rearranging to encode end expressa functional human heavy chain variable region and capable oftrans-switching (e.g., having a cis-linked RSS); optionally furthercomprising (3) a human light chain (e.g., kappa) transgene capable ofrearranging to encode a functional human light chain variable region andexpressing a human sequence light chain; optionally further comprising(4) a homozygous functionally disrupted endogenous light chain locus (κ,preferably κ and λ); and optionally further comprising (5) a serumcomprising an antibody comprising a chimeric heavy chain composed of ahuman sequence variable region encoded by a human transgene and a murineconstant region sequence encoded by an endogenous murine heavy chainconstant region gene (e.g., γ1, γ2a, γ2b, γ3).

Such transgenic mice may further comprise a serum comprising chimericantibodies which bind a predetermined human antigen (e.g., CD4, CD8,CEA) with an affinity of about at least 1×10⁴ M⁻¹, preferably with anaffinity of about at least 5×10⁴ M⁻¹, more preferably with an affinityof at least 1×10⁷ M⁻¹ to 1×10⁹ M⁻¹ or more. Frequently, hybridomas canbe made wherein the monoclonal antibodies produced thereby have anaffinity of at least 8×10⁷ M⁻¹. Chimeric antibodies comprising a heavychain composed of a murine constant region and a human variable region,often capable of binding to a nonhuman antigen, may also be present inthe serum or as an antibody secreted from a hybridoma.

In some variations, it is desirable to generate transgenic mice whichhave inactivated endogenous mouse heavy chain loci which retain intactheavy chain constant region genes, and which have a human heavy chaintransgene capable of trans-switching, and optionally also have a humanlight chain transgene, optionally with one or more inactivatedendogenous mouse light chain loci. Such mice may advantageously produceB cells capable of alternatively expressing antibodies comprising fullyhuman heavy chains and antibodies comprising chimeric (humanvariable/mouse constant) heavy chains, by trans-switching. The serum ofsaid mice would contain antibodies comprising fully human heavy chainsand antibodies comprising chimeric (human variable/mouse constant) heavychains, preferably in combination with fully human light chains.Hybridomas can be generated from the B cells of said mice.

Generally, such chimeric antibodies can be generated by trans-switching,wherein a human transgene encoding a human variable region (encoded byproductive V-D-J rearrangement in vivo) and a human constant region,typically human μ, undergoes switch recombination with a non-transgeneimmunoglobulin constant gene switch sequence (RSS) thereby operablylinking the transgene-encoded human variable region with a heavy chainconstant region which is not encoded by said transgene, typically anendogenous murine immunoglobulin heavy chain constant region or aheterologous (e.g., human) heavy chain constant region encoded on asecond transgene. Whereas cis-switching refers to isotype-switching byrecombination of RSS elements within a transgene, trans-switchinginvolves recombination between a transgene RSS and an RSS elementoutside the transgene, often on a different chromosome than thechromosome which harbors the transgene.

Trans-switching generally occurs between an RSS of an expressedtransgene heavy chain constant region gene and either an RSS of anendogenous murine constant region gene (of a non-μ isotype, typically γ)or an RSS of a human constant region gene contained on a secondtransgene, often integrated on a separate chromosome.

When trans-switching occurs between an RSS of a first, expressedtransgene heavy chain constant region gene (e.g., μ) and an RSS of ahuman heavy chain constant region gene contained on a second transgene,a non-chimeric antibody having a substantially fully human sequence isproduced. For example and not limitation, a polynucleotide encoding ahuman heavy chain constant region (e.g., γ1) and an operably linked RSS(e.g., a γ1 RSS) can be introduced (e.g., transfected) into a populationof hybridoma cells generated from a transgenic mouse B-cell (or B cellpopulation) expressing an antibody comprising a transgene-encoded humanμ chain. The resultant hybridoma cells can be selected for the presenceof the introduced polynucleotide and/or for the expression oftrans-switched antibody comprising a heavy chain having the variableregion (idiotype/antigen reactivity) of the human A chain and having theconstant region encoded by the introduced polynucleotide sequence (e.g.,human γ1). Trans-switch recombination between the RSS of thetransgene-encoded human A chain and the RSS of the introducedpolynucleotide encoding a downstream isotype (e.g., γ1) thereby cangenerate a trans-switched antibody.

The invention also provides a method for producing such chimerictrans-switched antibodies comprising the step of immunizing with apredetermined antigen a transgenic mouse comprising a genome comprising:(1) a homozygous functionally disrupted endogenous heavy chain locuscomprising at least one murine constant region gene capable oftrans-switching (e.g., γ2a, γ2b, γ1, γ3), (2) a human heavy chaintransgene capable of rearranging to encode a functional human heavychain variable region and expressing a human sequence heavy chain andcapable of undergoing isotype switching (and/or trans-switching), andoptionally further comprising (3) a human light chain (e.g., kappa)transgene capable of rearranging to encode a functional human light(e.g., kappa) chain variable region and expressing a human sequencelight chain, and optionally further comprising (4) a homozygousfunctionally disrupted endogenous light chain locus (typically κ,preferably both κ and λ), and optionally further comprising (5) a serumcomprising an antibody comprising a chimeric heavy chain composed of ahuman sequence variable region encoded by a human transgene and a murineconstant region sequence encoded by an endogenous murine heavy chainconstant region gene (e.g., γ1, γ2a, γ2b, γ3).

Affinity Tagging Selecting for Switched Isotypes

Advantageously, trans-switching (and cis-switching) is associated withthe process of somatic mutation. Somatic mutation expands the range ofantibody affinities encoded by clonal progeny of a B-cell. For example,antibodies produced by hybridoma cells which have undergone switching(trans- or cis-) represent a broader range of antigen-binding affinitiesthan is present in hybridoma cells which have not undergone switching.Thus, a hybridoma cell population (typically clonal) which expresses afirst antibody comprising a heavy chain comprising a first human heavychain variable region in polypeptide linkage to a first human heavychain constant region (e.g., μ) can be screened for hybridoma cellclonal variants which express an antibody comprising a heavy chaincontaining said first human heavy chain variable region in polypeptidelinkage to a second heavy chain constant region (e.g., a human γ, α, orε constant region). Such clonal variants can be produced by naturalclonal variation producing cis-switching in vitro, by induction of classswitching (trans- or cis-) as through the administration of agents thatpromote isotype switching, such as T-cell-derived lymphokines (e.g.,IL-4 and IFN_(γ)), by introduction of a polynucleotide comprising afunctional RSS and a heterologous (e.g. human) heavy chain constantregion gene to serve as a substrate for trans-switching, or by acombination of the above, and the like. Often, polynucleotidescontaining a human downstream isotype constant region (e.g., γ1, γ3, andthe like) with an operably linked RSS will also be introduced intohybridoma cells to promote isotype switching via the trans-switchmechanism.

Class switching and affinity maturation take place within the samepopulation of B cells derived from transgenic animals of the presentinvention. Therefore, identification of class-switched B cells (orhybridomas derived therefrom) can be used as a screening step forobtaining high affinity monoclonal antibodies. A variety of approachescan be employed to facilitate class switching events such ascis-switching (intratransgene switching), trans-switching, or both. Forexample, a single continuous human genomic fragment comprising both μand ε constant region genes with the associated RSS elements and switchregulatory elements (e.g., sterile transcript promoter) can be used as atransgene. However, some portions of the desired single contiguous humangenomic fragment can be difficult to clone efficiently, such as due toinstability problems when replicated in a cloning host or the like; inparticular, the region between δ and γ3 can prove difficult to cloneefficiently, especially as a contiguous fragment comprising the μ gene,γ3 gene, a V gene, D gene segments, and J gene segments.

Also for example, a discontinuous human transgene (minigene) composed ofa human μ gene, human γ3 gene, a human V gene(s), human D gene segments,and human J gene segments, with one or more deletions of an intervening(intronic) or otherwise nonessential sequence (e.g., one or more V, D,and/or J segment and/or one or more non-μ constant region gene(s)). Suchminigenes have several advantages as compared to isolating a singlecontiguous segment of genomic DNA spanning all of the essential elementsfor efficient immunoglobulin expression and switching. For example, sucha minigene avoids the necessity of isolating large pieces of DNA whichmay contain sequences which are difficult to clone (e.g., unstablesequences, poison sequences, and the like). Moreover, minilocicomprising elements necessary for isotype switching (e.g., human γsterile transcript promoter) for producing cis- or trans-switching, canadvantageously undergo somatic mutation and class switching in vivo. Asmany eukaryotic DNA sequences can prove difficult to clone, omittingnon-essential sequences can prove advantageous.

In a variation, hybridoma clones producing antibodies having highbinding affinity (e.g., at least 1×10⁷ M⁻¹, preferably at least 1×10⁸M⁻¹, more preferably at least 1×10⁹ M⁻¹ or greater) are obtained byselecting, from a pool of hybridoma cells derived from B cells oftransgenic mice harboring a human heavy chain transgene capable ofisotype switching (see, supra) and substantially lacking endogenousmurine heavy chain loci capable of undergoing productive (in-frame)V-D-J rearrangement, hybridomas which express an antibody comprising aheavy chain comprising a human sequence heavy chain variable region inpolypeptide linkage to a human (or mouse) non-μ heavy chain constantregion; said antibodys are termed “switched antibodies” as they comprisea “switched heavy chain” which is produced as a consequence ofcis-switching and/or trans-switching in vivo or in cell culture.Hybridomas producing switched antibodies generally have undergone theprocess of somatic mutation, and a pool of said hybridomas willgenerally have a broader range of antigen binding affinities from whichhybridoma clones secreting high affinity antibodies can be selected.Typically, hybridomas secreting a human sequence antibody havingsubstantial binding affinity (greater than 1×10⁷ M⁻¹ to 1×10⁸ M⁻¹) for apredetermined antigen and wherein said human sequence antibody compriseshuman immunoglobulin variable region(s) can be selected by a methodcomprising a two-step process. One step is to identify and isolatehybridoma cells which secrete immunoglobulins which comprise a switchedheavy chain (e.g., by binding hybridoma cells to an immobilizedimmunoglobulin which specifically binds a switched heavy chain and doesnot substantially bind to an unswitched isotype, e.g., μ). The otherstep is to identify hybridoma cells which bind to the predeterminedantigen with substantial binding affinity (e.g., by ELISA of hybridomaclone supernatants, FACS analysis using labeled antigen, and the like).Typically, selection of hybridomas which secrete switched antibodies isperformed prior to identifying hybridoma cells which bind predeterminedantigen. Hybridoma cells which express switched antibodies that havesubstantial binding affinity for the predetermined antigen are isolatedand cultured under suitable growth conditions known in the art,typically as individual selected clones. Optionally, the methodcomprises the step of culturing said selected clones under conditionssuitable for expression of monoclonal antibodies; said monoclonalantibodies are collected and can be administered for therapeutic,prophylactic, and/or diagnostic purposes.

Often, the selected hybridoma clones can serve as a source of DNA or RNAfor isolating immunoglobulin sequences which encode immunoglobulins(e.g. a variable region) that bind to (or confer binding to) thepredetermined antigen. Subsequently, the human variable region encodingsequence may be isolated (e.g., by PCR amplification or cDNA cloningfrom the source (hybridoma clone)) and spliced to a sequence encoding adesired human constant region to encode a human sequence antibody moresuitable for human therapeutic uses where immunogenicity is preferablyminimized. The polynucleotide(s) having the resultant fully humanencoding sequence(s) can be expressed in a host cell (e.g., from anexpression vector in a mammalian cell) and purified for pharmaceuticalformulation.

Xenoenhancers

A heterologous transgene capable of encoding a human immunoglobulin(e.g., a heavy chain) advantageously comprises a cis-linked enhancerwhich is not derived from the mouse genome, and/or which is notnaturally associated in cis with the exons of the heterologoustransgene. For example, a human κ transgene (e.g., a κ minilocus) canadvantageously comprise a human Vκ gene, a human Jκ gene, a human Cκgene, and a xenoenhancer, typically said xenoenhancer comprises a humanheavy chain intronic enhancer and/or a murine heavy chain intronicenhancer, typically located between a Jκ gene and the Cκ gene, orlocated downstream of the Cκ gene. For example, the mouse heavy chainJ-μ intronic enhancer (Banerji et al. (1983) Cell 33: 729) can beisolated on a 0.9 kb XbaI fragment of the plasmid pKVe2 (see, infra).The human heavy chain J-μ intronic enhancer (Hayday et al. (1984) Nature307: 334) can be isolated as a 1.4 kb MluI/HindIII fragment (see,infra). Addition of a transcriptionally active xenoenhancer to atransgene, such as a combined xenoenhancer consisting essentially of ahuman J-μ intronic enhancer linked in cis to a mouse J-μ intronicenhancer, can confer high levels of expression of the transgene,especially where said transgene encodes a light chain, such as human κ.Similarly, a rat 3′ enhancer can be advantageously included in aminilocus construct capable of encoding a human heavy chain.

Specific Preferred Embodiments

A preferred embodiment of the invention is an animal containing at leastone, typically 2-10, and sometimes 25-50 or more copies of the transgenedescribed in Example 12 (e.g., pHC1 or pHC2) bred with an animalcontaining a single copy of a light chain transgene described inExamples 5, 6, 8, or 14, and the offspring bred with the J_(H) deletedanimal described in Example 10. Animals are bred to homozygosity foreach of these three traits. Such animals have the following genotype: asingle copy (per haploid set of chromosomes) of a human heavy chainunrearranged mini-locus (described in Example 12), a single copy (perhaploid set of chromosomes) of a rearranged human κ light chainconstruct (described in Example 14), and a deletion at each endogenousmouse heavy chain locus that removes all of the functional J_(H)segments (described in Example 10). Such animals are bred with mice thatare homozygous for the deletion of the J_(H) segments (Examples 10) toproduce offspring that are homozygous for the J_(H) deletion andhemizygous for the human heavy and light chain constructs. The resultantanimals are injected with antigens and used for production of humanmonoclonal antibodies against these antigens.

B cells isolated from such an animal are monospecific with regard to thehuman heavy and light chains because they contain only a single copy ofeach gene. Furthermore, they will be monospecific with regards to humanor mouse heavy chains because both endogenous mouse heavy chain genecopies are nonfunctional by virtue of the deletion spanning the J_(H)region introduced as described in Example 9 and 12. Furthermore, asubstantial fraction of the B cells will be monospecific with regards tothe human or mouse light chains because expression of the single copy ofthe rearranged human κ light chain gene will allelically andisotypically exclude the rearrangement of the endogenous mouse κ and λchain genes in a significant fraction of B-cells.

The transgenic mouse of the preferred embodiment will exhibitimmunoglobulin production with a significant repertoire, ideallysubstantially similar to that of a native mouse. Thus, for example, inembodiments where the endogenous Ig genes have been inactivated, thetotal immunoglobulin levels will range from about 0.1 to 10 mg/ml ofserum, preferably 0.5 to 5 mg/ml, ideally at least about 1.0 mg/ml. Whena transgene capable of effecting a switch to IgG from IgM has beenintroduced into the transgenic mouse, the adult mouse ratio of serum IgGto IgM is preferably about 10:1. Of course, the IgG to IgM ratio will bemuch lower in the immature mouse. In general, greater than about 10%,preferably 40 to 80% of the spleen and lymph node B cells expressexclusively human IgG protein.

The repertoire will ideally approximate that shown in a non-transgenicmouse, usually at least about 10% as high, preferably 25 to 50% or more.Generally, at least about a thousand different immunoglobulins (ideallyIgG), preferably 10⁴ to 10⁶ or more, will be produced, dependingprimarily on the number of different V, J and D regions introduced intothe mouse genome. These immunoglobulins will typically recognize aboutone-half or more of highly antigenic proteins, including, but notlimited to: pigeon cytochrome C, chicken lysozyme, pokeweed mitogen,bovine serum albumin, keyhole limpit hemocyanin, influenzahemagglutinin, staphylococcus protein A, sperm whale myoglobin,influenza neuraminidase, and lambda repressor protein. Some of theimmunoglobulins will exhibit an affinity for preselected antigens of atleast about 10⁷ M⁻¹, preferably 10⁸ M⁻¹ to 10⁹ M⁻¹ or greater.

In some embodiments, it may be preferable to generate mice withpredetermined repertoires to limit the selection of V genes representedin the antibody response to a predetermined antigen type. A heavy chaintransgene having a predetermined repertoire may comprise, for example,human V_(H) genes which are preferentially used in antibody responses tothe predetermined antigen type in humans. Alternatively, some V_(H)genes may be excluded from a defined repertoire for various reasons(e.g., have a low likelihood of encoding high affinity V regions for thepredetermined antigen; have a low propensity to undergo somatic mutationand affinity sharpening; or are immunogenic to certain humans).

Thus, prior to rearrangement of a transgene containing various heavy orlight chain gene segments, such gene segments may be readily identified,e.g. by hybridization or DNA sequencing, as being from a species oforganism other than the transgenic animal.

The transgenic mice of the present invention can be immunized with apredetermined antigen, such as a transmembrane proteins, cell surfacemacromolecule, or other suitable antigen (e.g., TNF, LPS, etc.) forwhich a human antibody would be desirable. The mice will produce B cellswhich undergo class-switching via intratransgene switch recombination(cis-switching) and express immunoglobulins reactive with thepredetermined-antigen. The immunoglobulins can be human sequenceantibodies, wherein the heavy and light chain polypeptides are encodedby human transgene sequences, which may include sequences derived bysomatic mutation and V region recombinatorial joints, as well asgermline-encoded sequences; these human sequence immunoglobulins can bereferred to as being substantially identical to a polypeptide sequenceencoded by a human V_(L) or V_(H) gene segment and a human J_(L) orJ_(L) segment, even though other non-germline sequences may be presentas a result of somatic mutation and differential V-J and V-D-Jrecombination joints. With respect to such human sequence antibodies,the variable regions of each chain are typically at least 80 percentencoded by human germline V, J, and, in the case of heavy chains, D,gene segments; frequently at least 85 percent of the variable regionsare encoded by i human germline sequences present on the transgene;often 90 or 95 percent or more of the variable region sequences areencoded by human germline sequences present on the transgene. However,since non-germline sequences are introduced by somatic mutation and VJand VDJ joining, the human sequence antibodies will frequently have somevariable region sequences (and less frequently constant regionsequences) which are not encoded by human V, D, or J gene segments asfound in the human transgene(s) in the germline of the mice. Typically,such non-germline sequences (or individual nucleotide positions) willcluster in or near CDRs, or in regions where somatic mutations are knownto cluster.

The human sequence antibodies which bind to the predetermined antigencan result from isotype switching, such that human antibodies comprisinga human sequence y chain (such as γ1, γ2a, γ2B, or γ3) and a humansequence light chain (such as K) are produced. Such isotype-switchedhuman sequence antibodies often contain one or more somatic mutation(s),typically in the variable region and often in or within about 10residues of a CDR) as a result of affinity maturation and selection of Bcells by antigen, particularly subsequent to secondary (or subsequent)antigen challenge. These high affinity human sequence antibodies mayhave binding affinities of at least 1×10⁹ M⁻¹, typically at least 5×10⁹M⁻¹, frequently more than 1×10¹⁰ M⁻¹, and sometimes 5×10¹⁰ M⁻¹ to1×10⁻¹¹ or greater. Such high affinity human sequence antibodies can bemade with high binding affinities for human antigens, such as human CD4and the like human macromolecules (e.g., such as a human transmembraneor cell surface protein or other cell surface antigen).

The B cells from such mice can be used to generate hybridomas expressingmonoclonal high affinity (greater than 2×10⁹ M⁻¹) human sequenceantibodies against a variety of antigens, including human proteins suchas CD4 and the like. These hybridomas can be used to generate acomposition comprising an immunoglobulin having an affinity constant(K_(a)) of at least 2×10⁹ M⁻¹ for binding to a predetermined humanantigen, wherein said immunoglobulin consists of:

a human sequence light chain composed of (1) a light chain variableregion having a polypeptide sequence which is substantially identical toa polypeptide sequence encoded by a human V_(L) gene segment and a humanJ_(L) segment, and (2) a light chain constant region having apolypeptide sequence which is substantially identical to a polypeptidesequence encoded by a human C_(L) gene segment; and

a human sequence heavy chain composed of a (1) a heavy chain variableregion having a polypeptide sequence which is substantially identical toa polypeptide sequence encoded by a human V_(H) gene segment, optionallya D region, and a human J_(H) segment, and (2) a constant region havinga polypeptide sequence which is substantially identical to a polypeptidesequence encoded by a human C_(H) gene segment.

Often, the human sequence heavy chain and human sequence light chain areseparately encoded by a human heavy chain transgene and a human lightchain transgene, respectively, which are integrated into a mouse cellgenome. However, both chains may be encoded on a single transgene, orone or both chains may be encoded on multiple transgenes, such as ahuman heavy chain transgene (e.g., HC2) which derived a V gene segmentfrom a YAC containing a V_(H) array which is not integrated are the samelocus as the human heavy chain transgene in the mouse germline.

In one embodiment, the composition has an immunoglobulin which comprisesa human sequence light chain having a κ constant region and a humansequence heavy chain having a γ constant region.

The mice (and hybridomas derived therefrom) are a source for animmunoglobulin having an affinity constant (K_(a)) of at least 1×10¹⁰M⁻¹ for binding to a predetermined human antigen, wherein saidimmunoglobulin consists of:

a human sequence light chain composed of (1) a light chain variableregion having a polypeptide sequence which is substantially identical toa polypeptide sequence encoded by a human V_(L) gene segment and a humanJ_(L) segment, and (2) a light chain constant region having apolypeptide sequence which is substantially identical to a polypeptidesequence encoded by a human C_(L) gene segment; and

a human sequence heavy chain composed of a (1) a heavy chain variableregion having a polypeptide sequence which is substantially identical toa-polypeptide sequence encoded by a human V_(H) gene segment, optionallya D region, and a human J_(H) segment, and (2) a constant region havinga polypeptide sequence which is substantially identical to a polypeptidesequence encoded by a human C_(H) gene segment.

The invention provides a transgenic mouse comprising: a homozygous pairof functionally disrupted endogenous heavy chain alleles, a homozygouspair of functionally disrupted endogenous light chain alleles, at leastone copy of a heterologous immunoglobulin light chain transgene, and atleast one copy of a heterologous immunoglobulin heavy chain transgene,and wherein said animal makes an antibody response followingimmunization with a human antigen wherein the antibody responsecomprises an immunoglobulin having an affinity constant (K_(a)) of atleast 2×10⁹ M⁻¹ for binding to a predetermined human antigen, whereinsaid immunoglobulin consists of:

a human sequence light chain composed of (1) a light chain variableregion having a polypeptide sequence which is substantially identical toa polypeptide sequence encoded by a human V_(L) gene segment and a humanJ_(L) segment, and (2) a light chain constant region having apolypeptide sequence which is substantially identical to a polypeptidesequence encoded by a human C_(L) gene segment; and

a human sequence heavy chain composed of a (1) a heavy chain variableregion having a polypeptide sequence which is substantially identical toa polypeptide sequence encoded by a human V_(H) gene segment, optionallya D region, and a human J_(H) segment, and (2) a constant region havinga polypeptide sequence which is substantially identical to a polypeptidesequence encoded by a human C_(H) gene segment.

Such a transgenic mouse can produce a human sequence immunoglobulinwhich binds to a human surface or transmembrane protein present on atleast one somatic cell type of a human, wherein the immunoglobulin bindssaid human surface or transmembrane protein with an affinity constant(K_(a)) of between 1.5×10⁹ M⁻¹ and 1.8×10¹⁰ M⁻¹. One example of such ahuman surface or transmembrane protein is CD4, although others may beused as immunogens as desired.

The development of high affinity human sequence antibodies againstpredetermined antigens is facilitated by a method for expanding therepertoire of human variable region gene segments in a transgenic mousehaving a genome comprising an integrated human immunoglobulin transgene,said method comprising introducing into the genome a V gene transgenecomprising V region gene segments which are not present in saidintegrated human immunoglobulin transgene. Often, the V region transgeneis a yeast artificial chromosome comprising a portion of a human V_(H)or V_(L) (V_(κ)) gene segment array, as may naturally occur in a humangenome or as may be spliced together separately by recombinant methods,which may include out-of-order or omitted V gene segments. Often atleast five or more functional V gene segments are contained on the YAC.In this variation, it is possible to make a transgenic mouse produced bythe V repertoire expansion method, wherein the mouse expresses animmunoglobulin chain comprising a variable region sequence encoded by aV region gene segment present on the V region transgene and a C regionencoded on the human Ig transgene. By means of the V repertoireexpansion method, transgenic mice having at least 5 distinct V genes canbe generated; as can mice containing at least about 24 V genes or more.Of course, some V gene segments may be non-functional (e.g., pseudogenesand the like); these segments may be retained or may be selectivelydeleted by recombinant methods available to the skilled artisan, ifdesired.

Once the mouse germline has been engineered to contain a functional YAChaving an expanded V segment repertoire, substantially not present inthe human Ig transgene containing the J and C gene segments, the traitcan be propagated and bred into other genetic backgrounds, includingbackgrounds where the functional YAC having an expanded V segmentrepertoire is bred into a mouse germline having a different human Igtransgene. Multiple functional YACs having an expanded V segmentrepertoire may be bred into a germline to work with a human Ig transgene(or multiple human Ig transgenes). Although referred to herein as YACtransgenes, such transgenes when integrated into the genome maysubstantially lack yeast sequences, such as sequences required forautonomous replication in yeast; such sequences may optionally beremoved by genetic engineering (e.g., restriction digestion andpulsed-field gel electrophoresis or other suitable method) afterreplication in yeast in no longer necessary (i.e., prior to introductioninto a mouse ES cell or mouse prozygote).

The invention also provides a method of propagating the trait of humansequence immunoglobulin expression, comprising breeding a transgenicmouse having the human Ig transgene(s), and optionally also having afunctional YAC having an expanded V segment repertoire. Both V_(H) andV_(L) gene segments may be present on the YAC. The transgenic mouse maybe bred into any background desired by the practitioner, includingbackgrounds harboring other human transgenes, including human Igtransgenes and/or transgenes encoding other human lymphocyte proteins.

The invention also provides a high affinity human sequenceimmunoglobulin produced by a transgenic mouse having an expanded Vregion repertoire YAC transgene.

Although the foregoing describes a preferred embodiment of thetransgenic animal of the invention, other embodiments are defined by thedisclosure herein and more particularly by the transgenes described inthe Examples. Four categories of transgenic animal may be defined:

I. Transgenic animals containing an unrearranged heavy and rearrangedlight immunoglobulin transgene.

II. Transgenic animals containing an unrearranged heavy and unrearrangedlight immunoglobulin transgene

III. Transgenic animal containing rearranged heavy and an unrearrangedlight immunoglobulin transgene, and

IV. Transgenic animals containing rearranged heavy and rearranged lightimmunoglobulin transgenes.

Of these categories of transgenic animal, the preferred order ofpreference is as follows II>I>III>IV where the endogenous light chaingenes (or at least the κ gene) have been knocked out by homologousrecombination (or other method) and I>II>III>IV where the endogenouslight chain genes have not been knocked out and must be dominated byallelic exclusion.

As is discussed supra, the invention provides human sequence monoclonalantibodies that are useful in treatment of human diseases. Therapeuticuses of monoclonal antibodies are discussed in, e.g., Larrick andBourla, Journal of Biological Response Modifiers, 5:379-393, which isincorporated herein by reference. Uses of human monoclonal antibodiesinclude treatment of autoimmune diseases, cancer, infectious diseases,transplant rejection, blood disorders such as coagulation disorders, andother diseases.

The antibodies of this invention may be administered to patients by anymethod known in the medical arts for delivery of proteins. Antibodiesare particularly suited for parenteral administration (i.e,subcutaneous, intramuscular or intravenous administration). Thepharmaceutical compositions of the present invention are suitable foradministration using alternative drug delivery approaches as well (see,e.g., Langer, Science, 249:1527-1533 (1990)).

Pharmaceutical compositions for parenteral administration usuallycomprise a solution of a monoclonal antibody dissolved in an acceptablecarrier, preferably an aqueous carrier. A variety of aqueous carrierscan be used, e.g., water, buffered water, 0.4% saline, 0.3% glycine andthe like. These solutions are sterile and generally free of particulatematter. These compositions may be sterilized by conventional, well knownsterilization techniques. The compositions may contain pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions such as pH-adjusting and buffering agents, tonicity adjustingagents and the like, for example sodium acetate, sodium chloride,potassium chloride, calcium chloride, sodium lactate, etc. Theconcentration of antibody in these formulations can vary widely, i.e.,from less than about 0.5%, usually at or at least about 0.1% to as muchas 1.5% or 2.0% by weight and will be selected primarily based on fluidvolumes, viscosities, etc., in accordance with the particular mode ofadministration selected. Actual methods for preparing parenterallyadministrable compositions will be known or apparent to those skilled inthe art and are described in more detail in, for example, Remington'sPharmaceutical Sciences, 17th Ed., Mack Publishing Company, Easton, Pa.(1985), which is incorporated herein by reference.

The compositions containing the present antibodies or a cocktail thereofcan be administered for the prophylactic and/or therapeutic treatments.In therapeutic application, compositions are administered to a patientin an amount sufficient to cure or at least partially arrest theinfection and its complications. An amount adequate to accomplish thisis defined as a “therapeutically effective dose.” Amounts effective forthis use generally range from about 0.05 mg/kg body weight to about 5mg/kg body weight, preferably between about 0.2 mg/kg body weight toabout 1.5 mg/kg body weight.

In some instances it will be desirable to modify the immunoglobulinmolecules of the invention to change their biological activity. Forexample, the immunoglobulins can be directly or indirectly coupled toother chemotherapeutics agent. A variety of chemotherapeutics can becoupled for targeting. For example, anti-inflammatory agents which maybe coupled include immunomodulators, platelet activating factor (PAF)antagonists, cyclooxygenase inhibitors, lipoxygenase inhibitors, andleukotriene antagonists. Some preferred moieties include cyclosporin A,indomethacin, naproxen, FK-506, mycophenolic acid, and the like.Similarly, anti-oxidants, e.g., superoxide dismutase, are useful intreating reperfusion injury. Likewise, anticancer agents, such asdaunomycin, doxorubicin, vinblastine, bleomycin, and the like can betargeted.

The monoclonal antibodies of the invention may also be used to targetamphipaths (e.g., liposomes) to sites in a patient. In thesepreparations, the drug to be delivered is incorporated as part of aliposome in which a human monoclonal antibody is embedded.

The human-sequence monoclonal antibodies of the invention are useful, inpart, because they bind specifically to the predetermined antigenagainst which they are directed. When the predetermined antigen is ahuman antigen (i.e., a | human protein or fragment thereof), it willsometimes be advantageous if the human immunoglobulin of the inventionalso binds to the cognate antigen found in non-human animals, especiallyanimals that are used frequently for drug testing (e.g., preclinicaltesting of biological activity, pharmacokinetics and safety). Theseanimals include mice, rabbits, rats, dogs, pigs, and, especially,non-human primates such as chimpanzees, apes and monkeys (e.g., Rhesusmonkeys and cynomolgus monkeys). The ability to recognize antigens inexperimental animals-is particularly useful for determining the effectof specific binding on biodistribution of the immunoglobulins. A cognateantigen is an antigen that (i) has a structure (e.g., amino acidsequence) that is substantially similar to the human antigen (i.e., theamino acid sequence of an animal cognate protein will typically be atleast about 50% identical to the human protein, usually at least about70% identical and often at least about 80% identical or more); (ii) hassubstantially the same function as the human antigen; and, (iii) oftenis found in the same cellular compartment as the human antigen. Humanand animal cognate antigens typically (but not always) have the samenames. Examples of cognate antigens include human tubulin and mousetubulin, human CD4 and Rhesus CD4, and human IgG and Rat IgG.

An other aspect, the invention provides antigen-binding human mABscomprising at least one polypeptide encoded by an artificial gene. Anartificial gene comprises a polypeptide-encoding nucleic acid segmentthat is synthesized in vitro by chemical or enzymatic methods that donot require a cell-derived template nucleic acid strand (e.g., a nucleicacid template obtained from a bacterial cell or an immune or hybridomacell) and the progeny (through replication) of the artificial gene,i.e., a wholly synthetic nucleic acid.

Although it is routine in genetic engineering to use short syntheticnucleic acids as primers, linkers and the like, it is also possible bychemical and/or enzymatic means to produce wholly syntheticprotein-coding nucleic acids that are 30, 50, or more bases in length.The artificial genes of the invention may include both synthetic nucleicacid regions and cell-derived nucleic acid regions. The syntheticnucleic acid region of the artificial gene will generally be at leastabout 50 bases in length, often at least about 100 bases, typically atleast about 200 bases, more often at least about 250 bases and usuallyover 300 bases or 400 bases in length. Typically the synthetic nucleicacid regions will encode variable gene segments or a portion thereof,e.g., CDR regions, and the constant regions will be encoded bycell-derived nucleic acids. Immunoglobulin polypeptides (i.e.,immunoglobulin heavy chains and immunoglobulin light chains) can beconveniently expressed using artificial genes that encode thepolypeptides. Usually the artificial genes are operably linked totranscription promoter sequences, e.g., promoter sequences derived fromimmunoglobulin genes or from viruses (e.g., SV40, CMV, HIV, RSV) orhybrid promoters. The artificial gene may be linked to other sequencesas well, e.g. polyadenylation sequences and introns. One method forexpressing an immunoglobulin polypeptide involves insertion of asynthetic nucleic acid encoding one region of an immunoglobulinpolypeptide (e.g., a variable region or portion thereof) into a vectorthat encodes the remaining segments or parts of the immunoglobulin chain(e.g., a μ, γ, γ2, γ3, γ4, δ, ε, α₁, or α₂ constant region) and,optionally, promoter (e.g., a CMV (cytomegalovirus) promoter),polyadenylation or other sequences. Such vectors are constructed so thatupon introduction into a cell, the cellular transcription andtranslation of the vector sequences results in an immunoglobinpolypeptide.

Functional human sequence immunoglobulin heavy and light chain genes andpolypeptides can be constructed using artificial genes, and used toproduce immunoglobulins with a desired specificity such as specificbinding to a predetermined antigen. This is accomplished by constructingan artificial gene that encodes an immunoglobulin polypeptidesubstantially similar to a polypeptide expressed by a cell from, or ahybridoma derived from, a transgenic animal immunized with thepredetermined antigen. Thus, the invention provides artificial genesencoding immunoglobulin polypeptides and methods for producing ahuman-sequence immunoglobulin using an artificial gene(s).

According to this method, a transgenic animal (e.g., a transgenic mousewith a homozygous pair of functionally disrupted endogenous heavy chainalleles, a homozygous pair of functionally disrupted endogenous lightchain alleles, at least one copy of a human immunoglobulin light chaintransgene, and at least one copy of a human immunoglobulin heavy chaintransgene) is immunized-with predetermined antigen, e.g., a humanprotein. Nucleic acid, preferably mRNA, is then collected or isolatedfrom a cell or population of cells in which immunoglobulin generearrangement has taken place, and the sequence(s) of nucleic acidsencoding the heavy and/or light chains (especially the V segments) ofimmunoglobulins, or a portion thereof, is determined. This sequenceinformation is used as a basis for the sequence of the artificial gene.

Sequence determination will generally require isolation of at least aportion of the gene or cDNA of interest, e.g., a portion of a rearrangedhuman transgene or corresponding cDNA encoding an immunoglobulinpolypeptide. Usually this requires cloning the DNA or, preferably, mRNA(i.e., cDNA) encoding the human immunoglobulin polypeptide. Cloning iscarried out using standard techniques (see, e.g., Sambrook et al. (1989)Molecular Cloning: A Laboratory Guide, Vols 1-3, Cold Spring HarborPress, which is incorporated herein by reference). For example, a cDNAlibrary may be constructed by reverse transcription of polyA+ mRNA,preferably membrane-associated mRNA, and the library screened usingprobes specific for human immunoglobulin polypeptide gene sequences. Ina preferred embodiment, however, the polymerase chain reaction (PCR) isused to amplify cDNAs (or portions of full-length cDNAs) encoding animmunoglobulin gene segment of interest (e.g., a light chain variablesegment). Because the sequences of the human immunoglobulin polypeptidegenes are readily available to those of skill, probes or PCR primersthat will specifically hybridize to or amplify a human immunoglobulingene or segment thereof can be easily designed. See, e.g., Taylor etal., Nuc. Acids. Res., 20:6287 (1992) which is incorporated byreference. Moreover, the sequences of the human transgene of thetransgenic mouse will often be known to the practicioner, and primersequences can be chosen that hybridize to appropriate regions of thetransgene. The amplified sequences can be readily cloned into anysuitable vector, e.g., expression vectors, minigene vectors, or phagedisplay vectors. It will be appreciated that the particular method ofcloning used not critical, so long as it is possible to determine thesequence of some portion of the immunoglobulin polypeptide of interest.As used herein, a nucleic acid that is cloned, amplified, tagged, orotherwise distinguished from background nucleic acids such that thesequence of the nucleic acid of interest can be determined, isconsidered isolated.

One source for RNA used for cloning and sequencing is a hybridomaproduced by obtaining a B cell from the transgenic mouse and fusing theB cell to an immortal cell. An advantage of using hybridomas is thatthey can be easily screened, and a hybridoma that produces a humanmonoclonal antibody of interest selected. Alternatively, RNA can beisolated from B cells (or whole spleen) of the immunized animal. Whensources other than hybridomas are used, it may be desirable to screenfor sequences encoding immunoglobulins or immunoglobulin polypeptideswith specific binding characteristics. One method for such screening isthe use of phage display technology. Phage display is described in e.g.,Dower et al., WO 91/17271, McCafferty et al., WO 92/01047, and Caton andKoprowski, Proc. Natl. Acad. Sci. USA, 87:6450-6454 (1990), each ofwhich is incorporated herein by reference. In one embodiment using phagedisplay technology, cDNA from an immunized transgenic mouse (e.g., totalspleen cDNA) is isolated, the polymerase chain reaction is used toamplify a cDNA sequences that encode a portion of an immunoglobulinpolypeptide, e.g., CDR regions, and the amplified sequences are insertedinto a phage vector. cDNAs encoding peptides of interest, e.g., variableregion peptides with desired binding characteristics, are identified bystandard techniques such as panning.

The sequence of the amplified or cloned nucleic acid is then determined.Typically the sequence encoding an entire variable region of theimmunoglobulin polypeptide is determined, however, it will sometimes byadequate to sequence only a portion of a variable region, for example,the CDR-encoding portion. Typically the portion sequenced will be atleast 30 bases in length, more often based coding for at least aboutone-third or aty least about one-half of the length of the variableregion will be sequenced.

Sequencing can be carried on clones isolated from a cDNA library, or,when PCR is used, after subcloning the amplified sequence or by directPCR sequencing of the amplified segment. Sequencing is carried out usingstandard techniques (see, e.g., Sambrook et al. (1989) MolecularCloning: A Laboratory Guide, Vols 1-3, Cold Spring Harbor Press, andSanger, F. et al. (1977) Proc. Natl. Acad. Sci. USA 74: 5463-5467, whichis incorporated herein by reference). By comparing the sequence of thecloned nucleic acid with published sequences of human immunoglobulingenes and cDNAs, one of skill will readily be able to determine,depending on the region sequenced, (i) the germline segment usage of thehybridoma immunoglobulin polypeptide (including the isotype of the heavychain) and (ii) the sequence of the heavy and light chain variableregions, including sequences resulting from N-region addition and theprocess of somatic mutation. One source of immunoglobulin gene sequenceinformation is the National Center for Biotechnology Information,National Library of Medicine, National Institutes of Health, Bethesda,Md.

In an alternative embodiment, the amino acid sequence of animmunoglobulin of interest may be determined by direct proteinsequencing.

An artificial gene can be constructed that has a sequence identical toor substantially similar to, at least a portion of theimmunoglobulin-expressing gene (i.e., rearranged transgene). Similarly,the artificial gene can encode an polypeptide that is identical or hassubstantial similarity to a polypeptide encoded by the sequenced portionof the rearranged transgene. The degeneracy of the genetic code allowsthe same polypeptide to be encoded by multiple nucleic acid sequences.It is sometimes desirable to change the nucleic acid sequence, forexample to introduce restriction sites, change codon usage to reflect aparticular expression system, or to remove a glycosylation site. Inaddition, changes in the hybridoma sequences may be introduced to changethe characteristics (e.g., binding characteristics) of theimmunoglobulin. For example, changes may be introduced, especially inthe CDR regions of the heavy and light chain variable regions, toincrease the affinity of the immunoglobulin for the predeterminedantigen.

Methods for constructing an synthetic nucleic acids are well known. Anentirely chemical synthesis is possible but in general, a mixedchemical-enzymatic synthesis is carried out in which chemicallysynthesized oligonucleotides are used in ligation reactions and/or inthe polymerase chain reaction to create longer polynucleotides. In amost preferred embodiment, the polymerase chain reaction is carried outusing overlapping primers chosen so that the result of the amplificationis a DNA with the sequence desired for the artificial gene. Theoligonucleotides of the present invention may be synthesized in solidphase or in solution. Generally, solid phase synthesis is preferred.Detailed descriptions of the procedures for solid phase synthesis ofoligonucleotides by phosphite-triester, phosphotriester, andH-phosphonate chemistries are widely available. See, for example,Itakura, U.S. Pat. No. 4,401,796; Caruthers et al., U.S. Pat. Nos.4,458,066 and 4,500,707; Beaucage et al., Tetrahedron Lett.,22:1859-1862; Matteucci et al., J. Amer. Chem. Soc., 103:3185-3191(1981); Caruthers et al., Genetic Engineering, 4:1-17 (1982); Jones,chapter 2, Atkinson et al., chapter 3, and Sproat et al., chapter 4, inGait, ed. Oligonucleotide Synthesis: A Practical Approach, IRL Press,Washington, D.C. (1984); Froehler et al., Tetrahedron Lett., 27:469-472(1986); Froehler et al., Nucleic Acids Res., 14:5399-5407 (1986); Sinhaet al., Tetrahedron Lett., 24:5843-5846 (1983); and Sinha et al.,Nucleic Acids Res., 12:4539-4557 (1984) which are incorporated herein byreference.

The artificial gene can introduced into a cell and expressed to producean immunoglobulin polypeptide. The choice of cell type for expressionwill depend on many factors (e.g., the level of protein glycosylationdesired), but cells capable of secreting human immunoglobulins will bepreferred. Especially preferred cells include CHO cells andmyeloma-derived cells such as the SP20 and NS0 cell lines. Standard cellculture are well known and are also described in Newman, et al.,Biotechnology, 10:1455-1460 (1992); Bebbington, et al., Biotechnology,10:169-175 (1992); Cockett, et al., Biotechnology, 8:662-667 (1990);Carter, et al., Biotechnology, 10:163-167 (1992), each of which isincorporated herein by reference. Methods for introduction of nucleicacids, e.g., an artificial gene, are well known and include transfection(e.g., by electroporation or liposome-mediated) and transformation.Systems for expression of introduced genes are described generally inSambrook et al., supra.

It is often desirable to express two immunoglobulin polypeptides (i.e.,a heavy chain and a light chain) in the same cell so that animmunoglobulin (e.g., an IgG molecule) is produced in vivo. Accordinglyit will sometimes be desirable to introduce two artificial genes (i.e.,one encoding a heavy chain and one encoding a light chain) into a cell.(The two artificial genes can be introduced on a single vector).Alternatively, one artificial gene encoding one immunoglobulinpolypeptide can be introduced into a cell that has been geneticallyengineered to express the other immunoglobulin polypeptide.

It will be apparent that as the cells into which the artificial gene istransfected propagate, the wholly synthetic nucleic acid portion of theartificial gene, will act as a template for replication andtranscription. Nonetheless, the progeny genes will have originated froma synthetic nucleic acid (i.e., a polypeptide-encoding nucleic acidmolecule that is synthesized in vitro by chemical or enzymatic methodsthat do not require a cell-derived template nucleic acid strand) and asused herein, are also considered artificial genes. Thus, therelationship of the synthetic portion of the artificial gene to theexpressed transgene of the hybridoma is one in which there is aninformational link (i.e., sequence information) but no direct physicallink.

The invention also provides anti-CD4 monoclonal antibodies useful intherapeutic and diagnostic applications, especially the treatment ofhuman disease. CD4 is a cell surface protein that is expressed primarilyon thymocytes and T cells, and which is involved in T-cell function andMHC Class II recognition of antigen. Antibodies directed against CD4 actto reduce the activity of CD4 cells and thus reduce undesirableautoimmune reactions, inflammatory responses and rejection oftransplanted organs.

The ability of a human anti-CD4 mAb to inhibit a T-helper cell dependentimmune response in primates can be demonstrated by immunizing theprimate with a soluble foreign antigen (e.g., tetanus toxoid (TT)) andmeasuring the ability of the primate to mount a delayed-typehypersensitivity reaction (DTH) to the antigen (e.g., followinginjection of the human mAb). The DTH is mediated by CD4⁺ (T-helper)cells (E. Benjamin and S. Lescowitz, Immunology: A Short Course, SecondEdition, (1991) Wiley-Liss, Inc., New York, pp. 277-292).Antigen-specific T-helper cells recognize the processed antigenpresented by MHC Class II molecules on antigen-presenting cells andbecome activated. The activated T-helper cells secrete a variety oflymphokines (IL2, INFγ, TNFβ, MCF) and thus attract and activatemacrophages and T-cytotoxic cells at the injection site. Although mostof the effector functions occurring as part of the DTH are performed bymacrophages and T-cytotoxic cells, it is the T-helper cells whichinitiate-the response. Therefore, if the T-helper cells can beinhibited, there will be no DTH. Administration of anti-CD4 mABs hasbeen shown to prevent (Wofsy, et al., J. Exp. Med., 161:378-391 (1985))or reverse (Wofsy, et al., J. Immunol., 138:3247-3253 (1987), Waldor, etal., Science, 227:415-417 (1985)) autoimmune disease in animal models.Administration of murine or chimeric anti-CD4 mAbs to patients withrheumatoid arthritis has shown evidence of clinical benefit (Knox, etal., Blood, 77:20-30 (1991); Goldbery, et al., J. Autoimmunity,4:617-630; Herzog, et al., Lancet, ii:1461-1462; Horneff, et al.,Arthritis Rheum., 34:129-140; Reiter, et al., Arthritis Rheum.,34:525-536; Wending, et al., J. Rheum., 18:325-327; Van der Lubbe, etal., Arthritis Rheum., 38:1097-1106; Van der Lubbe, et al., ArthritisRheum., 36:1375-1379; Moreland, et al., Arthritis Rheum., 36:307-318,and Choy, et al., Arthritis and Rheumatism, 39(1):52-56 (1996); all ofwhich is incorporated herein by reference). In addition, as noted above,a chimeric anti-CD4 mAB has shown some clinical efficacy in patientswith mycosis fungoides (Knox et al. (1991) Blood 77:20; which isincorporated herein by reference). Anti-CD4 antibodies are alsodiscussed in Newman, et al., Biotechnology, 10:1455-1460 (1992), whichis incorporated herein by reference.

The invention also provides anti-interleukin-8 monoclonal antibodiesuseful in therapeutic and diagnostic applications, especially thetreatment of human diseases. Interleukin-8 (IL8), a very potent andmostly specific chemoattractant for neutrophils, is thought to play animportant role in the inflammatory response. IL8 also inducesangiogenesis, mediates transendothelial neutrophil migration andcontributes to other inflammatory responses. The properties of IL8 andrelated cytokines are discussed in Baggiolini et al., 1994, Adv.Immunol. 55:97-179, which is incorporated herein by reference.

IL8 has been shown to bind to, and activate, neutrophils and to induceneutrophil chemotaxis through an endothelial cell layer in vitro. Therole of IL8 in inducing neutrophil transmigration from the vasculatureto a site of inflammation has been demonstrated in vivo as well.Moreover, inhibition of the IL8 in those circumstances has preventedtissue damage resulting from neutrophil recruitment. Anti-rabbit IL8antibodies can prevent lung reperfusion injuries resulting from ischemia(Sekido et al., 1993, Nature 365:654-7). Anti-rabbit IL8 antibodies canalso prevent lung damage resulting from endotoxin-induced pleurisy inrabbits (Broaddus et al., 1994, J. Immunol. 152:2960-7). In vivo primatemodels are also suitable for determining the effects of anti-human IL8mAbs on migration of neutrophils from the vasculature to theinflammatory site. It has been shown that intradermal injection ofrhesus monkeys with endotoxin upregulates IL8 expression at theinjection site and ultimately results in neutrophil localization (Silberet al., 1994, Lab. Invest. 70:163-75).

Anti-IL8 antibodies have also been shown to reduce tissue damage andprolong survival in animal models of acute inflammation including adultrespiratory distress syndrome (ARDS) and acid induced lung injury(Sekido et al., 1993, Nature 365:654-7; Mulligan et al., 1993, J.Immunol. 150:5585-95; Broaddus et al., 1994, J. Immunol. 152:2960-7, allof which are incorporated herein by reference). Consistent with the roleof IL-8 in inflammation, human anti-IL-8 monoclonal antibodies can beused for treatment of a variety of conditions caused or aggravated by aninflammatory response, including reperfusion injuries (especially to thelung and heart), vasculitis, septic shock, autoimmune diseases(including glomerulonephritis, inflammatory bowel disease, rheumatoidarthritis and psoriasis), allergic reactions (e.g., asthma) and cysticfibrosis.

Two distinct IL8 receptors, IL8RA and IL8RB, have been identified(Holmes et al., 1991, Science 253:1278-80; Murphy et al., 1991, Science253:1280-3). Both receptors bind IL8, but only IL8RB binds other CXCchemokines. Both receptors are found in approximately equal numbers onneutrophils and some lymphocytes.

EXPERIMENTAL EXAMPLES Methods and Materials

Transgenic mice are derived according to Hogan, et al., “Manipulatingthe Mouse Embryo: A Laboratory Manual”, Cold Spring Harbor Laboratory,which is incorporated herein by reference.

Embryonic stem cells are manipulated according to published procedures(Teratocarcinomas and embryonic stem cells: a practical approach, E. J.Robertson, ed., IRL Press, Washington, D.C., 1987; Zjilstra et al.,Nature 342:435-438 (1989); and Schwartzberg et al., Science 246:799-803(1989), each of which is incorporated herein by reference).

DNA cloning procedures are carried out according to J. Sambrook, et al.in Molecular Cloning: A Laboratory Manual, 2d ed., 1989, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., which is incorporatedherein by reference.

Oligonucleotides are synthesized on an Applied Bio Systemsoligonucleotide synthesizer according to specifications provided by themanufacturer.

Hybridoma cells and antibodies are manipulated according to “Antibodies:A Laboratory Manual”, Ed Harlow and David Lane, Cold Spring HarborLaboratory (1988), which is incorporated herein by reference.

Example 1 Genomic Heavy Chain Human Ig Transgene

This Example describes the cloning and microinjection of a human genomicheavy chain immunoglobulin transgene which is microinjected into amurine zygote.

Nuclei are isolated from fresh human placental tissue as described byMarzluff et al., “Transcription and Translation: A Practical Approach”,B. D. Hammes and S. J. Higgins, eds., pp. 89-129, IRL Press, Oxford(1985)). The isolated nuclei (or PBS washed human spermatocytes) areembedded in a low melting point agarose matrix and lysed with EDTA andproteinase κ to expose high molecular weight DNA, which is then digestedin-the agarose with the restriction enzyme NotI as described by M.Finney in Current Protocols in Molecular Biology (F. Ausubel, et al.,eds. John Wiley & Sons, Supp. 4, 1988, Section 2.5.1).

The NotI digested DNA is then fractionated by pulsed field gelelectrophoresis as described by Anand et al., Nucl. Acids Res.17:3425-3433 (1989). Fractions enriched for the NotI fragment areassayed by Southern hybridization to detect one or more of the sequencesencoded by this fragment. Such sequences include the heavy chain Dsegments, J segments, μ and γ1 constant regions together withrepresentatives of all 6 VH families (although this fragment isidentified as 670 kb fragment from HeLa cells by Berman et al. (1988),supra., we have found it to be as 830 kb fragment from human placentalan sperm DNA). Those fractions containing this NotI fragment (see FIG.4) are pooled and cloned into the NotI site of the vector pYACNN inYeast cells. Plasmid pYACNN is prepared by digestion-of pYAC-4 Neo (Cooket al., Nucleic Acids Res. 16: 11817 (1988)) with EcoRI and ligation inthe presence of the oligonucleotide 5′-AAT TGC GGC CGC-3′ (SEQ ID NO.:25).

YAC clones containing the heavy chain NotI fragment are isolated asdescribed by Brownstein et al., Science 244:1348-1351 (1989), and Greenet al., Proc. Natl. Acad. Sci. USA 87:1213-1217 (1990), which areincorporated herein by reference. The cloned NotI insert is isolatedfrom high molecular weight yeast DNA by pulse field gel electrophoresisas described by M. Finney, op cit. The DNA is condensed by the additionof 1 mM spermine and microinjected directly into the nucleus of singlecell embryos previously described.

Example 2 Genomic κ Light Chain Human Ig Transgene Formed by In VivoHomologous Recombination

A map of the human K light chain has been described in Lorenz et al.,Nucl. Acids Res. 15:9667-9677 (1987), which is incorporated herein byreference.

A 450 kb XhoI to NotI fragment that includes all of C_(κ), the 3′enhancer, all J segments, and at least five different V segments isisolated and microinjected into the nucleus of single cell embryos asdescribed in Example 1.

Example 3 Genomic κ Light Chain Human Ig Transgene Formed by In VivoHomologous Recombination

A 750 kb MluI to NotI fragment that includes all of the above plus atleast 20 more V segments is isolated as described in Example 1 anddigested with BssHII to produce a fragment of about 400 kb.

The 450 kb XhoI to NotI fragment plus the approximately 400 kb MluI toBssHII fragment have sequence overlap defined by the BssHII and XhoIrestriction sites. Homologous recombination of these two fragments uponmicroinjection of a mouse zygote results in a transgene containing atleast an additional 15-20 V segments over that found in the 450 kbXhoI/NotI fragment (Example 2).

Example 4 Construction of Heavy Chain Mini-Locus

A. Construction of pGP1 and pGP2

pBR322 is digested with EcoRI and StyI and ligated with the followingoligonucleotides to generate pGP1 which contains a 147 base pair insertcontaining the restriction sites shown in FIG. 8. The generaloverlapping of these oligos is also shown in FIG. 9.

The oligonucleotides are:

oligo-1 SEQ ID NO.: (26) 5′-CTT GAG CCC GCC TAA TGA GCG GGC TTT TTT TTGCAT ACT GCG GCC-3′ oligo-2 SEQ ID NO.: (27) 5′-GCA ATG GCC TGG ATC CATGGC GCG CTA GCA TCG ATA TCT AGA GCT CGA GCA-3′ oligo-3 SEQ ID NO.: (28)5′-TGC AGA TCT GAA TTC CCG GGT ACC AAG CTT ACG CGT ACT AGT GCG GCCGCT-3′ oligo-4 SEQ ID NO.: (29) 5′-AAT TAG CGG CCG CAC TAG TAC GCG TAAGCT TGG TAC CCG GGA ATT-3′ oligo-5 SEQ ID NO.: (30) 5′-CAG ATC TGC ATGCTC GAG CTC TAG ATA TCG ATG CTA GCG CGC CAT GGA TCC-3′ oligo-6 SEQ IDNO.: (31) 5′-AGG CCA TTG CGG CCG CAG TAT GCA AAA AAA AGC CCG CTC ATT AGGCGG GCT-3′.

This plasmid contains a large polylinker flanked by rare cutting NotIsites for building large inserts that can be isolated from vectorsequences for microinjection. The plasmid is based on pBR322 which isrelatively low copy compared to the pUC based plasmids (pGP1 retains thepBR322 copy number control region near the origin of replication). Lowcopy number reduces the potential toxicity of insert sequences. Inaddition, PGP1 contains a strong transcription terminator sequencederived from trpA (Christie et al., Proc. Natl. Acad. Sci. USA 78:4180(1981)) inserted between the ampicillin resistance gene and thepolylinker. This further reduces the toxicity associated with certaininserts by preventing readthrough transcription coming from theampicillin promoters.

Plasmid pGP2 is derived from pGP1 to introduce an additional restrictionsite (SfiI) in the polylinker. pGP1 is digested with MluI and SpeI tocut the recognition sequences in the polylinker portion of the plasmid.

The following adapter oligonucleotides are ligated to the thus digestedpGP1 to form pGP2.

5′ CGC GTG GCC GCA ATG GCC A 3′ (SEQ. ID No.: 32) 5′ CTA GTG GCC ATT GCGGCC A 3′ (SEQ. ID No.: 33)

pGP2 is identical to pGP1 except that it contains an additional Sfi Isite located between the MluI and SpeI sites. This allows inserts to becompletely excised with SfiI as well as with NotI.

B. Construction of pRE3 (Rat Enhancer 3′)

An enhancer sequence located downstream of the rat constant region isincluded in the heavy chain constructs.

The heavy chain region 3′ enhancer described by Petterson et al., Nature344:165-168 (1990), which is incorporated herein by reference) isisolated and cloned. The rat IGH 3′ enhancer sequence is PCR amplifiedby using the following oligonucleotides:

(SEQ ID NO.: 34) 5′ CAG GAT CCA GAT ATC AGT ACC TGA AAC AGG GCT TGC 3′(SEQ ID NO.: 35) 5′ GAG CAT GCA CAG GAC CTG GAG CAC ACA CAG CCT TCC 3′

The thus formed double stranded DNA encoding the 3′ enhancer is cut withBamHI and SphI and clone into BamHI/SphI cut pGP2 to yield pRE3 (ratenhancer 3′).

C. Cloning of Human J-μ Region

A substantial portion of this region is cloned by combining two or morefragments isolated from phage lambda inserts. See FIG. 9.

A 6.3 kb BamHI/HindIII fragment that includes all human J segments(Matsuda et al., EMBO J., 7:1047-1051 (1988); Ravetech et al.m Cell,27:583-591 (1981), which are incorporated herein by reference) isisolated from human genomic DNA library using the oligonucleotide GGACTG TGT CCC TGT GTG ATG CTT TTG ATG TCT GGG GCC AAG (SEQ ID NO.: 36).

An adjacent 10 kb HindIII/BamII fragment that contains enhancer, switchand constant region coding exons (Yasui et al., Eur. J. Immunol.19:1399-1403 (1989)) is similarly isolated using the oligonucleotide:CAC CAA GTT GAC CTG CCT GGT CAC AGA CCT GAC CAC CTA TGA (SEQ ID NO.:37).

An adjacent 3′ 1.5 kb BamHI fragment is similarly isolated using clonepMUM insert as probe (pMUM is 4 kb EcoRI/HindIII fragment isolated fromhuman genomic DNA library with oligonucleotide:

(SEQ ID NO.: 38) CCT GTG GAC CAC CGC CTC CAC CTT CAT CGT CCT CTT CCTCCT.mu membrane exon 1) and cloned into pUC19.

pGP1 is digested with BamHI and BgIII followed by treatment with calfintestinal alkaline phosphatase.

Fragments (a) and (b) from FIG. 9 are cloned in the digested pGP1. Aclone is then isolated which is oriented such that 5′ BamHI site isdestroyed by BamHI/Bgl fusion. It is identified as pMU (see FIG. 10).pMU is digested with BamHI and fragment (c) from FIG. 9 is inserted. Theorientation is checked with HindIII digest. The resultant plasmid pHIG1(FIG. 10) contains an 18 kb insert encoding J and Cμ segments.

D. Cloning of Cμ Region

pGP1 is digested with BamHI and HindIII is followed by treatment withcalf intestinal alkaline phosphatase (FIG. 14). The so treated fragment(b) of FIG. 14 and fragment (c) of FIG. 14 are cloned into theBamHI/HindIII cut pGP1. Proper orientation of fragment (c) is checked byHindIII digestion to form pCON1 containing a 12 kb insert encoding theCμ region.

Whereas pHIG1 contains J segments, switch and μ sequences in its 18 kbinsert with an SfiI 3′ site and a SpeI 5′ site in a polylinker flankedby NotI sites, will be used for rearranged VDJ segments. pCON1 isidentical except that it lacks the J region and contains only a 12 kbinsert. The use of pCON1 in the construction of fragment containingrearranged VDJ segments will be described hereinafter.

E. Cloning of γ-1 Constant Region (pREG2)

The cloning of the human γ-1 region is depicted in FIG. 16.

Yamamura et al., Proc. Natl. Acad. Sci. USA 83:2152-2156 (1986) reportedthe expression of membrane bound human γ-1 from a transgene constructthat had been partially deleted on integration. Their results indicatethat the 3′ BamHI site delineates a sequence that includes thetransmembrane rearranged and switched copy of the gamma gene with a V-Cintron of less than 5 kb. Therefore, in the unrearranged, unswitchedgene, the entire switch region is included in a sequence beginning lessthan 5 kb from the 5′ end of the first γ-1 constant exon. Therefore itis included in the 5′ 5.3 kb HindIII fragment (Ellison et al., NucleicAcids Res. 10:4071-4079 (1982), which is incorporated herein byreference). Takahashi et al., Cell 29: 671-679 (1982), which isincorporated herein by reference, also reports that this fragmentcontains the switch sequence, and this fragment together with the 7.7 kbHindIII to BamHI fragment must include all of the sequences we need forthe transgene construct. An intronic sequence is a nucleotide sequenceof at least 15 contiguous nucleotides that occurs in an intron of aspecified gene.

Phage clones containing the γ-1 region are identified and isolated usingthe following oligonucleotide which is specific for the third exon ofγ-I (CH3).

(SEQ ID NO.: 39) 5′ TGA GCC ACG AAG ACC CTG AGG TCA AGT TCA ACT GGT ACGTGG 3′.

A 7.7 kb HindIII to BgIII fragment (fragment (a) in FIG. 11) is clonedinto HindIII/BgIII cut pRE3 to form pREG1. The upstream 5.3 kb HindIIIfragment (fragment (b) in FIG. 11) is cloned into HindIII digested pREG1to form pREG2. Correct orientation is confirmed by BamHI/SpeI digestion.

F. Combining Cγ and Cμ

The previously described plasmid pHIG1 contains human J segments and theCμ constant region exons. To provide a transgene containing the Cμconstant region gene segments, pHIG1 was digested with SfiI (FIG. 10).The plasmid pREG2 was also digested with SfiI to produce a 13.5 kbinsert containing human Cγ exons and the rat 3′ enhancer sequence. Thesesequences were combined to produce the plasmid pHIG3′ (FIG. 12)containing the human J segments, the human Cμ constant region, the humanCγ1 constant region and the rat 3′ enhancer contained on a 31.5 kbinsert.

A second plasmid encoding human Cμ and human Cγ1 without J segments isconstructed by digesting pCON1 with SfiI and combining that with theSfiI fragment containing the human Cγ region and the rat 3′ enhancer bydigesting pREG2 with SfiI. The resultant plasmid, pCON (FIG. 12)contains a 26 kb NotI/SpeI insert containing human Cμ, human γ1 and therat 3′ enhancer sequence.

G. Cloning of D Segment

The strategy for cloning the human D segments is depicted in FIG. 13.Phage clones from the human genomic library containing D segments areidentified and isolated using probes specific for diversity regionsequences (Ichihara et al., EMBO J. 7:4141-4150 (1988)). The followingoligonucleotides are used:

DXP1: (SEQ ID NO.: 40) 5′-TGG TAT TAC TAT GGT TCG GGG AGT TAT TAT AACCAC AGT GTC-3′ DXP4: (SEQ ID NO.: 41) 5′-GCC TGA AAT GGA GCC TCA GGG CACAGT GGG CAC GGA CAC TGT-3′ DN4: (SEQ ID NO.: 42) 5′-GCA GGG AGG ACA TGTTTA GGA TCT GAG GCC GCA CCT GAC ACC-3′

A 5.2 kb XhoI fragment (fragment (b) in FIG. 13) containing DLR1, DXP1,DXP′1, and DA1 is isolated from a phage clone identified with oligoDXP1.

A 3.2 kb XbaI fragment (fragment (c) in FIG. 13) containing DXP4, DA4and DK4 is isolated from a phage clone identified with oligo DXP4.

Fragments (b), (c) and (d) from FIG. 13 are combined and cloned into theXbaI/XhoI site of pGP1 to form pHIG2 which contains a 10.6 kb insert.

This cloning is performed sequentially. First, the 5.2 kb fragment (b)in FIG. 13 and the 2.2 kb fragment (d) of FIG. 13 are treated with calfintestinal alkaline phosphatase and cloned into pGP1 digested with XhoIand XbaI. The resultant clones are screened with the 5.2 and 2.2 kbinsert. Half of those clones testing positive with the 5.2 and 2.2 kbinserts have the 5.2 kb insert in the proper orientation as determinedby BamHI digestion. The 3.2 kb XbaI fragment from FIG. 13 is then clonedinto this intermediate plasmid containing fragments (b) and (d) toform-pHIG2. This plasmid contains diversity segments cloned into thepolylinker with a unique 5′ SfiI site and unique 3′ SpeI site. Theentire polylinker is flanked by NotI sites.

H. Construction of Heavy Chain Minilocus

The following describes the construction of a human heavy chainmini-locus which contain one or more V segments.

An unrearranged V segment corresponding to that identified as the Vsegment contained in the hybridoma of Newkirk et al., J. Clin. Invest.81:1511-1518 (1988), which is incorporated herein by reference, isisolated using the following oligonucleotide:

(SEQ ID NO.: 43) 5′-GAT CCT GGT TTA GTT AAA GAG GAT TTT ATT CAC CCC TGTGTC-3′

A restriction map of the unrearranged V segment is determined toidentify unique restriction sites which provide upon digestion a DNAfragment having a length approximately 2 kb containing the unrearrangedV segment together with 5′ and 3′ flanking sequences. The 5′ primesequences will include promoter and other regulatory sequences whereasthe 3′ flanking sequence provides recombination sequences necessary forV-DJ joining. This approximately 3.0 kb V segment insert is cloned intothe polylinker of pGB2 to form pVH1.

pVH1 is digested with SfiI and the resultant fragment is cloned into theSfiI site of pHIG2 to form a pHIG5′. Since pHIG2 contains D segmentsonly, the resultant pHIG5′ plasmid contains a single V segment togetherwith D segments. The size of the insert contained in pHIG5 is 10.6 kbplus the size of the V segment insert.

The insert from pHIG5 is excised by digestion with NotI and SpeI andisolated. pHIG3′ which contains J, Cμ and cγ1 segments is digested withSpeI and NotI and the 3′ kb fragment containing such sequences and therat 3′ enhancer sequence is isolated. These two fragments are combinedand ligated into NotI digested pGP1 to produce pHIG which containsinsert encoding a V segment, nine D segments, six functional J segments,Cμ, Cγ and the rat 3′ enhancer. The size of this insert is approximately43 kb plus the size of the V segment insert.

I. Construction of Heavy Chain Minilocus by Homologous Recombination

As indicated in the previous section, the insert of pHIG isapproximately 43 to 45 kb when a single V segment is employed. Thisinsert size is at or near the limit of that which may be readily clonedinto plasmid vectors. In order to provide for the use of a greaternumber of V segments, the following describes in vivo homologousrecombination of overlapping DNA fragments which upon homologousrecombination within a zygote or ES cell form a transgene containing therat 3′ enhancer sequence, the human Cμ, the human Cγ1, human J segments,human D segments and a multiplicity of human V segments.

A 6.3 kb BamHI/HindIII fragment containing human J segments (seefragment (a) in FIG. 9) is cloned into MluI/SpeI digested pHIG5′ usingthe following adapters:

5′ GAT CCA AGC AGT 3′ (SEQ ID NO.: 44) 5′ CTA GAC TGC TTG 3′ (SEQ IDNO.: 45) 5′ CGC GTC GAA CTA 3′ (SEQ ID NO.: 46) 5′ AGC TTA GTT CGA 3′(SEQ ID NO.: 47)

The resultant is plasmid designated pHIG5′O (overlap). The insertcontained in this plasmid contains human V, D and J segments. When thesingle V segment from pVH1 is used, the size of this insert isapproximately 17 kb plus 2 kb. This insert is isolated and combined withthe insert from pHIG3′ which contains the human J, Cμ, γ1 and rat 3′enhancer sequences. Both inserts contain human J segments which providefor approximately 6.3 kb of overlap between the two DNA fragments. Whencoinjected into the mouse zygote, in vivo homologous recombinationoccurs generating a transgene equivalent to the insert contained inpHIG.

This approach provides for the addition of a multiplicity of V segmentsinto the transgene formed in vivo. For example, instead of incorporatinga single V segment into pHIG5′, a multiplicity of V segments containedon (1) isolated genomic DNA, (2) ligated DNA derived from genomic DNA,or (3) DNA encoding a synthetic V segment repertoire is cloned intopHIG2 at the SfiI site to generate pHIG5′ V_(N). The J segments fragment(a) of FIG. 9 is then cloned into pHIG5′ V_(N) and the insert isolated.This insert now contains a multiplicity of V segments and J segmentswhich overlap with the J segments contained on the insert isolated frompHIG3′. When cointroduced into the nucleus of a mouse zygote, homologousrecombination occurs to generate in vivo the transgene encoding multipleV segments and multiple J segments, multiple D segments, the Cμ region,the Cγ1 region (all from human) and the rat 3′ enhancer sequence.

Example 5 Construction of Light Chain Minilocus

A. Construction of pEμ1.

The construction of pEμ1 is depicted in FIG. 16. The mouse heavy chainenhancer is isolated on the XbaI to EcoRI 678 bp fragment (Banerji etal., Cell 33:729-740 (1983)) from phage clones using oligo:

(SEQ ID NO.: 48) 5′ GAA TGG GAG TGA GGC TCT CTC ATA CCC TAT TCA GAA CTGACT 3′

This Eμ fragment is cloned into EcoRV/XbaI digested pGP1 by blunt endfilling in EcoRI site. The resultant plasmid is designated pEmu1.

B. Construction of κ Light Chain Minilocus

The κ construct contains at least one human V_(κ) segment, all fivehuman J_(κ) segments, the human J-C_(κ) enhancer, human κ constantregion exon, and, ideally, the human 3′ κ enhancer (Meyer et al., EMBOJ. 8:1959-1964 (1989)). The κ enhancer in mouse is 9 kb downstream fromC_(κ). However, it is as yet unidentified in the human. In addition, theconstruct contains a copy of the mouse heavy chain J-Cμ enhancers.

The minilocus is constructed from four component fragments:

(a) A 16 kb SmaI fragment that contains the human C_(κ) exon and the 3′human enhancer by analogy with the mouse locus;

(b) A 5′ adjacent 5 kb SmaI fragment, which contains all five Jsegments;

(c) The mouse heavy chain intronic enhancer isolated from pEμ1 (thissequence is included to induce expression of the light chain constructas early as possible in B-cell development. Because the heavy chaingenes are transcribed earlier than the light chain genes, this heavychain enhancer is presumably active at an earlier stage than theintronic κ enhancer); and

(d) A fragment containing one or more V segments.

The preparation of this construct is as follows. Human placental DNA isdigested with SmaI and fractionated on agarose gel by electrophoresis.Similarly, human placental DNA is digested with BamHI and fractionatedby electrophoresis. The 16 kb fraction is isolated from the SmaIdigested gel and the 11 kb region is similarly isolated from the gelcontaining DNA digested with BamHI.

The 16 kb SmaI fraction is cloned into Lambda FIX II (Stratagene, LaJolla, Calif.) which has been digested with XhoI, treated with klenowfragment DNA polymerase to fill in the XhoI restriction digest product.Ligation of the 16 kb SmaI fraction destroys the SmaI sites and lasesXhoI sites intact.

The 11 kb BamHI fraction is cloned into λ EMBL3 (Strategene, La Jolla,Calif.) which is digested-with BamHI prior to cloning.

Clones from each library were probed with the Cκ specific oligo:

(SEQ ID NO.: 49) 5′ GAA CTG TGG CTG CAC CAT CTG TCT TCA TCT TCC CGC CATCTG 3′

A 16 kb XhoI insert that was subcloned into the XhoI cut pEμ1 so that Cκis adjacent to the SmaI site. The resultant plasmid was designatedpKap1.

The above Cκ specific oligonucleotide is used to probe the λ EMBL3/BamHIlibrary to identify an 11 kb clone. A 5 kb SmaI fragment (fragment (b)in FIG. 20) is subcloned and subsequently inserted into pKap1 digestedwith SmaI. Those plasmids containing the correct orientation of Jsegments, Cκ and the Eμ enhancer are designated pKap2.

One or more V_(κ) segments are thereafter subcloned into the MluI siteof pKap2 to yield the plasmid pKapH which encodes the human Vκ segments,the human Jκ segments, the human Cκ segments and the human Eμ enhancer.This insert is excised by digesting pKapH with NotI and purified byagarose gel electrophoresis. The thus purified insert is microinjectedinto the pronucleus of a mouse zygote as previously described.

C. Construction of κ Light Chain Minilocus by In Vivo HomologousRecombination

The 11 kb BamHI fragment is cloned into BamHI digested pGP1 such thatthe 3′ end is toward the SfiI site. The resultant plasmid is designatedpKAPint. One or more Vκ segments is inserted into the polylinker betweenthe BamHI and SpeI sites in pKAPint to form pKapHV. The insert of pKapHVis excised by digestion with NotI and purified. The insert from pKap2 isexcised by digestion with NotI and purified. Each of these fragmentscontain regions of homology in that the fragment from pKapHV contains a5 kb sequence of DNA that include the J_(κ) segments which issubstantially homologous to the 5 kb SmaI fragment contained in theinsert obtained from pKap2. As such, these inserts are capable ofhomologously recombining when microinjected into a mouse zygote to forma transgene encoding V_(κ), J_(κ) and C_(κ).

Example 6 Isolation of Genomic Clones Corresponding to Rearranged andExpressed Copies of Immunoglobulin κ Light Chain Genes

This example describes the cloning of immunoglobulin κ light chain genesfrom cultured cells that express an immunoglobulin of interest. Suchcells may contain multiple alleles of a given immunoglobulin gene. Forexample, a hybridoma might contain four copies of the κ light chaingene, two copies from the fusion partner cell line and two copies fromthe original B-cell expressing the immunoglobulin of interest. Of thesefour copies, only one encodes the immunoglobulin of interest, despitethe fact that several of them may be rearranged. The procedure describedin this example allows for the selective cloning of the expressed copyof the κ light chain.

A. Double Stranded cDNA

Cells from human hybridoma, or lymphoma, or other cell line thatsynthesizes either cell surface or secreted or both forms of IgM with aK light chain are used for the isolation of polyA+ RNA. The RNA is thenused for the synthesis of oligo dT primed cDNA using the enzyme reversetranscriptase (for general methods see, Goodspeed et al. (1989) Gene 76:1; Dunn et al. (1989) J. Biol. Chem. 264: 13057). The single strandedcDNA is then isolated and G residues are added to the 3′ end using theenzyme polynucleotide terminal transferase. The G-tailed single-strandedcDNA is then purified and used as template for second strand synthesis(catalyzed by the enzyme DNA polymerase) using the followingoligonucleotide as a primer:

(SEQ ID NO.: 50) 5′-GAG GTA CAC TGA CAT ACT GGC ATG CCC CCC CCC CCC-3′

The double stranded cDNA is isolated and used for determining thenucleotide sequence of the 5′ end of the mRNAs encoding the heavy andlight chains of the expressed immunoglobulin molecule. Genomic clones ofthese expressed genes are then isolated. The procedure for cloning theexpressed light chain gene is outlined in part B below.

B. Light Chain

The double stranded cDNA described in part A is denatured and used as atemplate for a third round of DNA synthesis using the followingoligonucleotide primer:

(SEQ ID NO.: 51) 5′-GTA CGC CAT ATC AGC TGG ATG AAG TCA TCA GAT GGC GGGAAG ATG AAG ACA GAT GGT GCA-3′

This primer contains sequences specific for the constant portion of theκ light chain message (TCA TCA GAT GGC GGG AAG ATG AAG ACA GAT GGTGCA)(52) as well as unique sequences that can be used as a primer forthe PCR amplification of the newly synthesized DNA strand (GTA CGC CATATC AGC TGG ATG AAG)(53). The sequence is amplified by PCR using thefollowing two oligonucleotide primers:

(SEQ ID NO.: 54) 5′-GAG GTA CAC TGA CAT ACT GGC ATG-3′ (SEQ ID NO.: 53)5′-GTA CGC CAT ATC AGC TGG ATG AAG-3′

The PCR amplified sequence is then purified by gel electrophoresis andused as template for dideoxy sequencing reactions using the followingoligonucleotide as a primer:

(SEQ ID NO.: 54) 5′-GAG GTA CAC TGA CAT ACT GGC ATG-3′

The first 42 nucleotides of sequence will then be used to synthesize aunique probe for isolating the gene from which immunoglobulin messagewas transcribed. This synthetic 42 nucleotide segment of DNA will bereferred to below as o-kappa.

A Southern blot of DNA, isolated from the Ig expressing cell line anddigested individually and in pairwise combinations with severaldifferent restriction endonucleases including SmaI, is then probed withthe 32-P labelled unique oligonucleotide o-kappa. A unique restrictionendonuclease site is identified upstream of the rearranged V segment.

DNA from the Ig expressing cell line is then cut with SmaI and secondenzyme (or BamHI or KpnI if there is SmaI site inside V segment). Anyresulting non-blunted ends are treated with the enzyme T4 DNA polymeraseto give blunt ended DNA molecules. Then add restriction site encodinglinkers (BamHI, EcoRI or XhoI depending on what site does not exist infragment) and cut with the corresponding linker enzyme to give DNAfragments with BamHI, EcoRI or XhoI ends. The DNA is then sizefractionated by agarose gel electrophoresis, and the fraction includingthe DNA fragment covering the expressed V segment is cloned into lambdaEMBL3 or Lambda FIX (Stratagene, La Jolla, Calif.). V segment containingclones are isolated using the unique probe o-kappa. DNA is isolated frompositive clones and subcloned into the polylinker of pKap1. Theresulting clone is called pRKL.

Example 7 Isolation of Genomic Clones Corresponding to ArrangedExpressed Copies of Immunoglobulin Heavy Chain μ Genes

This example describes the cloning of immunoglobulin heavy chain μ genesfrom cultured cells of expressed and immunoglobulin of interest. Theprocedure described in this example allows for the selective cloning ofthe expressed copy of a μ heavy chain gene.

Double-stranded cDNA is prepared and isolated as described hereinbefore. The double-stranded cDNA is denatured and used as a template fora third round of DNA synthesis using the following oligonucleotideprimer:

(SEQ ID NO.: 55) 5′-GTA CGC CAT ATC AGC TGG ATG AAG ACA GGA GAC GAG GGGGAA AAG GGT TGG GGC GGA TGC-3′

This primer contains sequences specific for the constant portion of theμ heavy chain message (ACA GGA GAC GAG GGG GAA AAG GGT TGG GGC GGA TGC)(SEQ ID NO.: 56) as well as unique sequences that can be used as aprimer for the PCR amplification of the newly synthesized DNA strand(GTA CGC CAT ATC AGC TGG ATG AAG) (SEQ ID NO.: 53). The sequence isamplified by PCR using the following two oligonucleotide primers:

(SEQ ID NO.: 54) 5′ GAG GTA CAC TGA CAT ACT GGC ATG-3′ (SEQ ID NO.: 57)5′-GTA CTC CAT ATC AGC TGG ATG AAG-3′

The PCR amplified sequence is then purified by gel electrophoresis andused as template for dideoxy sequencing reactions using the followingoligonucleotide as a primer:

(SEQ ID NO.: 54) 5′-GAG GTA CAC TGA CAT ACT GGC ATG-3′

The first 42 nucleotides of sequence are then used to synthesize aunique probe for isolating the gene from which immunoglobulin messagewas transcribed. This synthetic 42 nucleotide segment of DNA will bereferred to below as o-mu.

A Southern blot of DNA, isolated from the Ig expressing cell line anddigested individually and in pairwise combinations with severaldifferent restriction endonucleases including MluI (MluI is a rarecutting enzyme that cleaves between the J segment and mu CH1), is thenprobed with the 32-P labelled unique oligonucleotide o-mu. A uniquerestriction endonuclease site is identified upstream of the rearranged Vsegment.

DNA from the Ig expressing cell line is then cut with MluI and secondenzyme. MluI or SpeI adapter linkers are then ligated onto the ends andcut to convert the upstream site to MluI or SpeI. The DNA is then sizefractionated by agarose gel electrophoresis, and the fraction includingthe DNA fragment covering the expressed V segment is cloned directlyinto the plasmid pGPI. V segment containing clones are isolated usingthe unique probe o-mu, and the insert is subcloned into MluI orMluI/SpeI cut plasmid pCON2. The resulting plasmid is called pRMGH.

Example 8 Construction of Human κ Miniloci Transgenes Light ChainMinilocus

A human genomic DNA phage library was screened with kappa light chainspecific oligonucleotide probes and isolated clones spanning the J_(κ)-Cregion. A 5.7 kb ClaI/XhoI fragment containing J_(κ)1 together with a 13kb XhoI fragment containing J_(κ)2-5 and C_(κ) into pGP1d was cloned andused to create the plasmid pKcor. This plasmid contains J_(κ)1-5, thekappa intronic enhancer and C_(κ) together with 4.5 kb of 5′ and 9 kb of3′ flanking sequences. It also has a unique 5′ XhoI site for cloningV_(κ) segments and a unique 3′ SalI site for inserting additionalcis-acting regulatory sequences.

V Kappa Genes

A human genomic DNA phage library was screened with V_(κ) light chainspecific oligonucleotide probes and isolated clones containing humanV_(κ) segments. Functional V segments were identified by DNA sequenceanalysis. These clones contain TATA boxes, open reading frames encodingleader and variable peptides (including 2 cysteine residues), splicesequences, and recombination heptamer-12 bp spacer-nonamer sequences.Three of the clones were mapped and sequenced. Two of the clones, 65.5and 65.8 appear to be functional, they contain TATA boxes, open readingframes encoding leader and variable peptides (including 2 cysteineresidues), splice sequences, and recombination heptamer-12 bpspacer-nonamer sequences. The third clone, 65.4, appears to encode aV_(κ)I pseudogene as it contains a non-canonical recombination heptamer.

One of the functional clones, Vk 65-8, which encodes a VkIII familygene, was used to build a light chain minilocus construct.

pKC1

The kappa light chain minilocus transgene pKC1 (FIG. 32) was generatedby inserting a 7.5 kb XhoI/SalI fragment containing V_(κ) 65.8 into the5′ XhoI site of pKcor. The transgene insert was isolated by digestionwith NotI prior to injection.

The purified insert was microinjected into the pronuclei of fertilized(C57BL/6xCBA)F2 mouse embryos and transferred the surviving embryos intopseudopregnant females as described by Hogan et al. (in Methods ofManipulating the Mouse Embryo, 1986, Cold Spring Harbor Laboratory, NewYork). Mice that developed from injected embryos were analyzed for thepresence of transgene sequences by Southern blot analysis of tail DNA.Transgene copy number was estimated by band intensity relative tocontrol standards containing known quantities of cloned DNA. Serum wasisolated from these animals and assayed for the presence of transgeneencoded human Ig kappa protein by ELISA as described by Harlow and Lane(in Antibodies: A Laboratory Manual, 1988, Cold Spring HarborLaboratory, New York). Microtiter plate wells were coated with mousemonoclonal antibodies specific for human Ig kappa (clone 6E1, #0173,AMAC, Inc., Westbrook, Me.), human IgM (Clone AF6, #0285, AMAC, Inc.,Westbrook, Me.) and human IgG1 (clone JL512, #0280, AMAC, Inc.,Westbrook, Me.). Serum samples were serially diluted into the wells andthe presence of specific immunoglobulins detected with affinity isolatedalkaline phosphatase conjugated goat anti-human Ig (polyvalent) that hadbeen pre-adsorbed to minimize cross-reactivity with mouseimmunoglobulins.

FIG. 35 shows the results of an ELISA assay of serum from 8 mice (I.D.#676, 674, 673, 670, 666, 665, 664, and 496). The first seven of thesemice developed from embryos that were injected with the pKC1 transgeneinsert and the eighth mouse is derived from a mouse generated bymicroinjection of the pHC1 transgene (described previously). Two of theseven mice from KC1 injected embryos (I.D.#'s 666 and 664) did notcontain the transgene insert as assayed by DAN Southern blot analysis,and five of the mice (I.D.#'s 676, 674, 673, 670, and 665) contained thetransgene. All but one of the KC1 transgene positive animals expressdetectable levels of human. Ig kappa protein, and the singlenon-expressing animal appears to be a genetic mosaic on the basis of DNASouthern blot analysis. The pHC1 positive transgenic mouse expresseshuman IgM and IgG1 but not Ig kappa, demonstrating the specificity ofthe reagents used in the assay.

pKC2

The kappa light chain minilocus transgene pKC2 was generated byinserting an 8 kb XhoI/SalI fragment containing V_(κ) 65.5 into the 5′XhoI site of pKC1. The resulting transgene insert, which contains twoV_(κ) segments, was isolated prior to microinjection by digestion withNotI.

pKVe2

This construct is identical to pKC1 except that it includes 1.2 kb ofadditional sequence 5′ of J_(κ) and is missing 4.5 kb of sequence 3′ ofV_(κ) 65.8. In additional it contains a 0.9 kb XbaI fragment containingthe mouse heavy chain J-μ intronic enhancer (Banerji et al., Cell 33:729-740 (1983)) together with a 1.4 kb MluI/HindIII fragment containingthe human heavy chain J-μ intronic enhancer (Hayday et al., Nature 307:334-340 (1984)) inserted downstream. This construct tests thefeasibility of initiating early rearrangement of the light chainminilocus to effect allelic and isotypic exclusion. Analogous constructscan be generated with different enhancers, i.e., the mouse or rat 3′kappa or heavy chain enhancer (Meyer and Neuberger, EMBO J. 8: 1959-1964(1989); Petterson et al. Nature 344: 165-168 (1990), which areincorporated herein by reference).

Rearranged Light Chain Transgenes

A kappa light chain expression cassette was designed to reconstructfunctionally rearranged light chain genes that have been amplified byPCR from human B-cell DNA. The scheme is outlined in FIG. 33. PCRamplified light chain genes are cloned into the vector pK5nx thatincludes 3.7 kb of 5′ flanking sequences isolated from the kappa lightchain gene 65.5. The VJ segment fused to the 5′ transcriptionalsequences are then cloned into the unique XhoI site of the vector pK31sthat includes J_(κ)2-4, the J_(κ) intronic enhancer, C_(κ), and 9 kb ofdownstream sequences. The resulting plasmid contains a reconstructedfunctionally rearranged kappa light chain transgene that can be excisedwith NotI for microinjection into embryos. The plasmids also containunique SalI sites at the 3′ end for the insertion of additionalcis-acting regulatory sequences.

Two synthetic oligonucleotides (o-130, o-131) were used to amplifyrearranged kappa light chain genes from human spleen genomic DNA.Oligonucleotide o-131 (gga ccc aga (g,c)gg aac cat gga a(g,a)(g,a,t,c))is complementary to the 5′ region of V_(κ) III family light chain genesand overlaps the first ATC of the leader sequence. Oligonucleotide o-130(gtg caa tca att ctc gag ttt gac tac aga c) is complementary to asequence approximately 150 bp 3′ of J_(κ)1 and includes an XhoI site.These two oligonucleotides amplify a 0.7 kb DNA fragment from humanspleen DNA corresponding to rearranged V_(κ) III genes joined to J_(κ)1segments. The PCR amplified DNA was digested with NcoI and XhoI andcloned individual PCR products into the plasmid pNNO3. The DNA sequenceof 5 clones was determined and identified two with functional VJ joints(open reading frames). Additional functionally rearranged light chainclones are collected. The functionally rearranged clones can beindividually cloned into light chain expression cassette described above(FIG. 33). Transgenic mice generated with the rearranged light chainconstructs can be bred with heavy chain minilocus transgenics to producea strain of mice that express a spectrum of fully human antibodies inwhich all of the diversity of the primary repertoire is contributed bythe heavy chain. One source of light chain diversity can be from somaticmutation. Because not all light chains will be equivalent with respectto their ability to combine with a variety of different heavy chains,different strains of mice, each containing different light chainconstructs can be generated and tested. The advantage of this scheme, asopposed to the use of unrearranged light chain miniloci, is theincreased light chain allelic and isotypic exclusion that comes fromhaving the light chain ready to pair with a heavy chain as soon as heavychain VDJ joining occurs. This combination can result in an increasedfrequency of B-cells expressing fully human antibodies, and thus it canfacilitate the isolation of human Ig expressing hybridomas.

NotI inserts of plasmids pIGM1, pHC1, pIGG1, pKC1, and pKC2 wereisolated away from vector sequences by agarose gel electrophoresis. Thepurified inserts were microinjected into the pronuclei of fertilized(C57BL/6xCBA)F2 mouse embryos and transferred the surviving embryos intopseudopregnant females as described by Hogan et al. (Hogan et al.,Methods of Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory,New York (1986).

Example 9 Inactivation of the Mouse Kappa Light Chain Gene byHomologous. Recombination

This example describes the inactivation of the mouse endogenous kappalocus by homologous recombination in embryonic stem (ES) cells followedby introduction of the mutated gene into the mouse germ line byinjection of targeted ES cells bearing an inactivated kappa allele intoearly mouse embryos (blastocysts).

The strategy is to delete J_(κ) and C_(K) by homologous recombinationwith a vector containing DNA sequences homologous to the mouse kappalocus in which a 4.5 kb segment of the locus, spanning the J_(κ) geneand C_(K) segments, is deleted and replaced by the selectable markerneo.

Construction of the Kappa Targeting Vector

The plasmid pGEM7 (KJ1) contains the neomycin resistance gene (neo),used for drug selection of transfected ES cells, under thetranscriptional control of the mouse phosphoglycerate kinase (pgk)promoter (XbaI/TaqI fragment; Adra et al. (1987) Gene 60: 65) in thecloning vector pGEM-7Zf(+). The plasmid also includes a heterologouspolyadenylation site for the neo gene, derived from the 3′ region of themouse pgk gene (PvuII/HindIII fragment; Boer et al., BiochemicalGenetics, 28: 299-308 (1990)). This plasmid was used as the startingpoint for construction of the kappa targeting vector. The first step wasto insert sequences homologous to the kappa locus 3′ of the neoexpression cassette.

Mouse kappa chain sequences (FIG. 20 a) were isolated from a genomicphage library derived from liver DNA using oligonucleotide probesspecific for the Cκ locus:

(SEQ ID NO.: 58) 5′-GGC TGA TGC TGC ACC AAC TGT ATC CAT CTT CCC ACC ATCCAG-3′and for the J_(K)5 gene segment:

(SEQ ID NO.: 59) 5′-CTC ACG TTC GGT GCT GGG ACC AAG CTG GAG CTG AAA CGTAAG-3′.

An 8 kb BglII/SacI fragment extending 3′ of the mouse C_(K) segment wasisolated from a positive phage clone in two pieces, as a 1.2 kbBglII/SacI fragment and a 6.8 kb SacI fragment, and subcloned intoBglII/SacI digested pGEM7 (KJ1) to generate the plasmid pNEO-K3′ (FIG.20 b).

A 1.2 kb EcoRI/SphI fragment extending 5′ of the J_(K) region was alsoisolated from a positive phage clone. An SphI/XbaI/BglII/EcoRI adaptorwas ligated to the SphI site of this fragment, and the resulting EcoRIfragment was ligated into EcoRI digested pNEO-K3′, in the same 5′ to 3′orientation as the neo gene and the downstream 3′ kappa sequences, togenerate pNEO-K5′3′ (FIG. 20 c).

The Herpes Simplex Virus (HSV) thymidine kinase (TK) gene was thenincluded in the construct in order to allow for enrichment of ES clonesbearing homologous recombinants, as described by Mansour et al., Nature336: 348-352 (1988), which is incorporated herein by reference. The HSVTK cassette was obtained from the plasmid pGEM7 (TK), which contains thestructural sequences for the HSV TK gene bracketed by the mouse pgkpromoter and polyadenylation sequences as described above for pGEM7(KJ1). The EcoRI site of pGEM7 (TK) was modified to a BamHI site and theTK cassette was then excised as a BamHI/HindIII fragment and subclonedinto pGP1b to generate pGP1b-TK. This plasmid was linearized at the XhoIsite and the XhoI fragment from pNEO-K5′3′, containing the neo geneflanked by genomic sequences from 5′ of Jκ and 3′ of Cκ, was insertedinto pGP1b-TK to generate the targeting vector J/C KI (FIG. 20 d). Theputative structure of the genomic kappa locus following homologousrecombination with J/C K1 is shown in FIG. 20 e.

Generation and Analysis of ES Cells with Targeted Inactivation of aKappa Allele

The ES cells used were the AB-1 line grown on mitotically inactiveSNL76/7 cell feeder layers (McMahon and Bradley, Cell 62: 1073-1085(1990)) essentially as described (Robertson, E. J. (1987) inTeratocarcinomas and Embryonic Stem Cells: A Practical Approach. E. J.Robertson, ed. (Oxford: IRL Press), p. 71-112). Other suitable ES linesinclude, but are not limited to, the E14 line (Hooper et al. (1987)Nature 326: 292-295), the D3 line (Doetschman et al. (1985) J. Embryol.Exp. Morph. 87: 27-45), and the CCE line (Robertson et al. (1986) Nature323: 445-448). The success of generating a mouse line from ES cellsbearing a specific targeted mutation depends on the pluripotence of theES cells (i.e., their ability, once injected into a host blastocyst, toparticipate in embryogenesis and contribute to the germ cells of theresulting animal).

The pluripotence of any given ES cell line can vary with time in cultureand the care with which it has been handled. The only definitive assayfor pluripotence is to determine whether the specific population of EScells to be used for targeting can give rise to chimeras capable ofgermline transmission of the ES genome. For this reason, prior to genetargeting, a portion of the parental population of AB-1 cells isinjected into C57Bl/6J blastocysts to ascertain whether the cells arecapable of generating chimeric mice with extensive ES cell contributionand whether the majority of these chimeras can transmit the ES genome toprogeny.

The kappa chain inactivation vector J/C K1 was digested with NotI andelectroporated into AB-1 cells by the methods described (Hasty et al.,Nature, 350: 243-246 (1991)). Electroporated cells were plated onto 100mm dishes at a density of 1-2×10⁶ cells/dish. After 24 hours, G418 (200μg/ml of active component) and FIAU (0.5 μM) were added to the medium,and drug-resistant clones were allowed to develop over 10-11 days.Clones were picked, trypsinized, divided into two portions, and furtherexpanded. Half of the cells derived from each clone were then frozen andthe other half analyzed for homologous recombination between vector andtarget sequences.

DNA analysis was carried out by Southern blot hybridization. DNA wasisolated from the clones as described (Laird et al., Nucl. Acids Res.19: 4293 (1991)) digested with XbaI and probed with the 800 bpEcoRI/XbaI fragment indicated in FIG. 20 e as probe A. This probedetects a 3.7 kb XbaI fragment in the wild type locus, and a diagnostic1.8 kb band in a locus which has homologously recombined with thetargeting vector (see FIGS. 20 a and e). Of 901 G418 and FIAU resistantclones screened by Southern blot analysis, 7 displayed the 1.8 kb XbaIband indicative of a homologous recombination into one of the kappagenes. These 7 clones were further digested with the enzymes BglII,SacI, and PstI to verify that the vector integrated homologously intoone of the kappa genes. When probed with the diagnostic 800 bpEcoRI/XbaI fragment (probe A), BglII, SacI, and PstI digests of wildtype DNA produce fragments of 4.1, 5.4, and 7 kb, respectively, whereasthe presence of a targeted kappa allele would be indicated by fragmentsof 2.4, 7.5, and 5.7 kb, respectively (see FIGS. 20 a and e). All 7positive clones detected by the XbaI digest showed the expected BglII,SacI, and PstI restriction fragments diagnostic of a homologousrecombination at the kappa light chain. In addition, Southern blotanalysis of an NsiI digest of the targeted clones using a neo specificprobe (probe B, FIG. 20 e) generated only the predicted fragment of 4.2kb, demonstrating that the clones each contained only a single copy ofthe targeting vector.

Generation of mice bearing the inactivated kappa chain. Five of thetargeted ES clones described in the previous section were thawed andinjected into C57Bl/6J blastocysts as described (Bradley, A. (1987) inTeratocarcinomas and Embryonic Stem Cells: A Practical Approach. E. J.Robertson, ed. (Oxford: IRL Press), p. 113-151) and transferred into theuteri of pseudopregnant females to generate chimeric mice resulting froma mixture of cells derived from the input ES cells and the hostblastocyst. The extent of ES cell contribution to the chimeras can bevisually estimated by the amount of agouti coat coloration, derived fromthe ES cell line, on the black C57Bl/6J background. Approximately halfof the offspring resulting from blastocyst injection of the targetedclones were chimeric (i.e., showed agouti as well as black pigmentation)and of these, the majority showed extensive (70 percent or greater) EScell contribution to coat pigmentation. The AB1 ES cells are an XY cellline and a majority of these high percentage chimeras were male due tosex conversion of female embryos colonized by male ES cells. Malechimeras derived from 4 of the 5 targeted clones were bred with C57BL/6Jfemales and the offspring monitored for the presence of the dominantagouti coat color indicative of germline transmission of the ES genome.Chimeras from two of these clones consistently generated agoutioffspring. Since only one copy of the kappa locus was targeted in theinjected ES clones, each agouti pup had a 50 percent chance ofinheriting the mutated locus. Screening for the targeted gene wascarried out by Southern blot analysis of Bgl II-digested DNA from tailbiopsies, using the probe utilized in identifying targeted ES clones(probe A, FIG. 20 e). As expected, approximately 50 percent of theagouti offspring showed a hybridizing Bgl II band of 2.4 kb in additionto the wild-type band of 4.1 kb, demonstrating the germline transmissionof the targeted kappa locus.

In order to generate mice homozygous for the mutation, heterozygoteswere bred together and the kappa genotype of the offspring determined asdescribed above. As expected, three genotypes were derived from theheterozygote matings: wild-type mice bearing two copies of a normalkappa locus, heterozygotes carrying one targeted copy of the kappa geneand one NT kappa gene, and mice homozygous for the kappa mutation. Thedeletion of kappa sequences from these latter mice was verified byhybridization of the Southern blots with a probe specific for J_(κ)(probe C, FIG. 20 a). Whereas hybridization of the J_(κ) probe wasobserved to DNA samples from heterozygous and wild-type siblings, nohybridizing signal was present in the homozygotes, attesting to thegeneration of a novel mouse strain in which both copies of the kappalocus have been inactivated by deletion as a result of targetedmutation.

Example 10 Inactivation of the Mouse Heavy Chain Gene by HomologousRecombination

This example describes the inactivation of the endogenous murineimmunoglobulin heavy chain locus by homologous recombination inembryonic stem (ES) cells. The strategy is to delete the endogenousheavy chain J segments by homologous recombination with a vectorcontaining heavy chain sequences from which the J_(H) region has beendeleted and replaced by the gene for the-selectable marker neo.

Construction of a Heavy Chain Targeting Vector

Mouse heavy chain sequences containing the J_(H) region (FIG. 21 a) wereisolated from a genomic phage library derived from the D3 ES cell line(Gossler et al., Proc. Natl. Acad. Sci. U.S.A. 83: 9065-9069 (1986))using a J_(H) 4 specific oligonucleotide probe:

(SEQ ID NO.: 60) 5′-ACT ATG CTA TGG ACT ACT GGG GTC AAG GAA CCT CAG TCACCG-3′

A 3.5 kb genomic SacI/StuI fragment, spanning the J_(H) region, wasisolated from a positive phage clone and subcloned into SacI/SmaIdigested pUC18. The resulting plasmid was designated pUC18 J_(H). Theneomycin resistance gene (neo), used for drug selection of transfectedES cells, was derived from a repaired version of the plasmid pGEM7(KJ1). A report in the literature (Yenofsky et al. (1990) Proc. Natl.Acad. Sci. (U.S.A.) 87: 3435-3439) documents a point mutation the neocoding sequences of several commonly used expression vectors, includingthe construct pMC1neo (Thomas and Cappechi (1987) Cell 51: 503-512)which served as the source of the neo gene used in pGEM7 (KJ1). Thismutation reduces the activity of the neo gene product and was repairedby replacing a restriction fragment encompassing the mutation with thecorresponding sequence from a wild-type neo clone. The HindIII site inthe prepared pGEM7 (KJ1) was converted to a SalI site by addition of asynthetic adaptor, and the neo expression cassette excised by digestionwith XbaI/SalI. The ends of the neo fragment were then blunted bytreatment with the Klenow form of DNA poll, and the neo fragment wassubcloned into the NaeI site of pUC18 J_(H), generating the plasmidpUC18 J_(H)-neo (FIG. 21 b).

Further construction of the targeting vector was carried out in aderivative of the plasmid pGP1b. pGP1b was digested with the restrictionenzyme NotI and ligated with the following oligonucleotide as anadaptor:

(SEQ ID NO.: 61) 5′-GGC CGC TCG ACG ATA GCC TCG AGG CTA TAA ATC TAG AAGAAT TCC AGC AAA GCT TTG GC-3′

The resulting plasmid, called pGMT, was used to build the mouseimmunoglobulin heavy chain targeting construct.

The Herpes Simplex Virus (HSV) thymidine kinase (TK) gene was includedin the construct in order to allow for enrichment of ES clones bearinghomologous recombinants, as described by Mansour et al. (Nature 336,348-352 (1988)). The HSV TK gene was obtained from the plasmid pGEM7(TK) by digestion with EcoRI and HindIII. The TK DNA fragment wassubcloned between the EcoRI and HindIII sites of pGMT, creating theplasmid pGMT-TK (FIG. 21 c).

To provide an extensive region of homology to the target sequence, a 5.9kb genomic XbaI/XhoI fragment, situated 5′ of the J_(H) region, wasderived from a positive genomic phage clone by limit digestion of theDNA with XhoI, and partial digestion with XbaI. As noted in FIG. 21 a,this XbaI site is not present in genomic DNA, but is rather derived fromphage sequences immediately flanking the cloned genomic heavy chaininsert in the positive phage clone. The fragment was subcloned intoXbaI/XhoI digested PGMT-TK, to generate the plasmid pGMT-TK-J_(H) 5′(FIG. 21 d).

The final step in the construction involved the excision from pUC18J_(H)-neo of the 2.8 kb EcoRI fragment which contained the neo gene andflanking genomic sequences 3′ of J_(H). This fragment was blunted byKlenow polymerase and subcloned into the similarly blunted XhoI site ofpGMT-TK-J_(H) 5′. The resulting construct, J_(H) KO1 (FIG. 21 e),contains 6.9 kb of genomic sequences flanking the J_(H) locus, with a2.3 kb deletion spanning the J_(H) region into which has been insertedthe neo gene. FIG. 21 f shows the structure of an endogenous heavy chaingene after homologous recombination with the targeting construct.

Example 11 Generation and Analysis of Targeted ES Cells

AB-1 ES cells (McMahon and Bradley, Cell 62: 1073-1085 (1990)) weregrown on mitotically inactive SNL76/7 cell feeder layers essentially asdescribed (Robertson, E. J. (1987) Teratocarcinomas and Embryonic StemCells: A Practical Approach. E. J. Robertson, ed. (Oxford: IRL Press),pp. 71-112). As described in the previous example, prior toelectroporation of ES cells with the targeting construct J_(H) KO1, thepluripotency of the ES cells was determined by generation of AB-1derived chimeras which were shown capable of germline transmission ofthe ES genome.

The heavy chain inactivation vector J_(H KO)1 was digested with NotI andelectroporated into AB-1 cells by the methods described (Hasty et al.,Nature 350: 243-246 (1991)). Electroporated cells were plated into 100mm dishes at a density of 1-2×10⁶ cells/dish. After 24 hours, G418 (200mg/ml of active component) and FIAU (0.5 mM) were added to the medium,and drug-resistant clones were allowed to develop over 8-10 days. Cloneswere picked, trypsinized, divided into two portions, and furtherexpanded. Half of the cells derived from each clone were then frozen andthe other half analyzed for homologous recombination between vector andtarget sequences.

DNA analysis was carried out by Southern blot hybridization. DNA wasisolated from the clones as described (Laird et al. (1991) Nucleic AcidsRes. 19: 4293), digested with StuI and probed with the 500 bp EcoRI/StuIfragment designated as probe A in FIG. 21 f. This probe detects a StuIfragment of 4.7 kb in the wild-type locus, whereas a 3 kb band isdiagnostic of homologous recombination of endogenous sequences with thetargeting vector (see FIGS. 21 a and f). Of 525 G418 and FIAUdoubly-resistant clones screened by Southern blot hybridization, 12 werefound to contain the 3 kb fragment diagnostic of recombination with thetargeting vector. That these clones represent the expected targetedevents at the J_(H) locus (as shown in FIG. 21 f) was confirmed byfurther digestion with HindIII, SpeI and HpaI. Hybridization of probe A(see FIG. 21 f) to Southern blots of HindIII, SpeI, and HpaI digestedDNA produces bands of 2.3 kb, >10 kb, and >10 kb, respectively, for thewild-type locus (see FIG. 21 a), whereas bands of 5.3 kb, 3.8 kb, and1.9 kb, respectively, are expected for the targeted heavy chain locus(see FIG. 21 f). All 12 positive clones detected by the StuI digestshowed the predicted HindIII, SpeI, and HpaI bands diagnostic of atargeted J_(H) gene. In addition, Southern blot analysis of a StuIdigest of all 12 clones using a neo-specific probe (probe B, FIG. 21_(f)) generated only the predicted fragment of 3 kb, demonstrating thatthe clones each contained only a single copy of the targeting vector.

Generation of Mice Carrying the J_(H) Deletion

Three of the targeted ES clones described in the previous section werethawed and injected into C57BL/6J blastocysts as described (Bradley, A.(1987) in Teratocarcinomas and Embryonic Stem Cells: A PracticalApproach, E. J. Robertson, ed. (Oxford: IRL Press), p. 113-151) andtransferred into the uteri of pseudopregnant females. The extent of EScell contribution to the chimera was visually estimated from the amountof agouti coat coloration, derived from the ES cell line, on the blackC57BL/6J background. Half of the offspring resulting from blastocystinjection of two of the targeted clones were chimeric (i.e., showedagouti as well as black pigmentation); the third targeted clone did notgenerate any chimeric animals. The majority of the chimeras showedsignificant (approximately 50 percent or greater) ES cell contributionto coat pigmentation. Since the AB-1 ES cells are an XY cell line, mostof the chimeras were male, due to sex conversion of female embryoscolonized by male ES cells. Males chimeras were bred with C57BL/6Jfemales and the offspring monitored for the presence of the dominantagouti coat color indicative of germline transmission of the ES genome.Chimeras from both of the clones consistently generated agoutioffspring. Since only one copy of the heavy chain locus was targeted inthe injected ES clones, each agouti pup had a 50 percent chance ofinheriting the mutated locus. Screening for the targeted gene wascarried out by Southern blot analysis of StuI-digested DNA from tailbiopsies, using the probe utilized in identifying targeted ES clones(probe A, FIG. 21 f). As expected, approximately 50 percent of theagouti offspring showed a hybridizing StuI band of approximately 3 kb inaddition to the wild-type band of 4.7 kb, demonstrating germlinetransmission of the targeted J_(H) gene segment.

In order to generate mice homozygous for the mutation, heterozygoteswere bred together and the heavy chain genotype of the offspringdetermined as described above. As expected, three genotypes were derivedfrom the heterozygote matings: wild-type mice bearing two copies of thenormal J_(H) locus, heterozygotes carrying one targeted copy of the geneand one normal copy, and mice homozygous for the J_(H) mutation. Theabsence of J_(H) sequences from these latter mice was verified byhybridization of the Southern blots of StuI-digested DNA with a probespecific for J_(H) (probe C, FIG. 21 a). Whereas hybridization of theJ_(H) probe to a 4.7 kb fragment in DNA samples from heterozygous andwild-type siblings was observed, no signal was present in samples fromthe J_(H)-mutant homozygotes, attesting to the generation of a novelmouse strain in which both copies of the heavy chain gene have beenmutated by deletion of the J_(H) sequences.

Example 12 Heavy Chain Minilocus Transgene

A. Construction of Plasmid Vectors for Cloning Large DNA Sequences

1. pGP1a

The plasmid pBR322 was digested with EcoRI and StyI and ligated with thefollowing oligonucleotides:

oligo-42 (SEQ ID NO.: 62) 5′-caa gag ccc gcc taa tga gcg ggc ttt ttt ttgcat act gcg gcc gct-3′ oligo-43 (SEQ ID NO.: 63) 5′-aat tag cgg ccg cagtat gca aaa aaa agc ccg ctc att agg cgg gct-3′

The resulting plasmid, pGP1a, is designed for cloning very large DNAconstructs that can be excised by the rare cutting restriction enzymeNotI. It contains a NotI restriction site downstream (relative to theampicillin resistance gene, AmpR) of a strong transcription terminationsignal derived from the trpA gene (Christie et al., Proc. Natl. Acad.Sci. USA 78: 4180 (1981)). This termination signal reduces the potentialtoxicity of coding sequences inserted into the NotI site by eliminatingreadthrough transcription from the AmpR gene. In addition, this plasmidis low copy relative to the pUC plasmids because it retains the pBR322copy number control region. The low copy number further reduces thepotential toxicity of insert sequences and reduces the selection againstlarge inserts due to DNA replication. The vectors pGP1b, pGP1c, pGP1d,and pGP1f are derived from pGP1a and contain different polylinkercloning sites. The polylinker sequences are given below

pGP1a        NotI      GCGGCCGC pGP1b (SEQ ID NO.: 64)   NotI  XhoI        ClaI         BamHI      HindIII  NotIGCggccgcctcgagatcactatcgattaattaaggatccagcagtaagcttgcGGCCGC pGI1c (SEQID NO. 65)    NotI    SmaI   XhoI  SalI  HindIII   BamHI SacII NotIGCggccgcatcccgggtctcgaggtcgacaagctttcgaggatccgcGGCCGC pGP1d (SEQ ID NO.66)    NotI   SalI HindIII ClaI BamHI XhoI   NotIGCggccgctgtcgacaagcttatcgatggatcctcgagtgcGGCCGC pGP1f (SEQ ID NO.67)   NotI   SalI HindIII EcoRI  ClaI     KpnI  BamHI XhoI    NotIGCggccgctgtcgacaagcttcgaattcagatcgatgtggtacctggatcctcgagtgcGGCCGCEach of these plasmids can be used for the construction of largetransgene inserts that are excisable with NotI so that the transgene DNAcan be purified away from vector sequences prior to microinjection.2. pGP1b pGP1a was digested with NotI and ligated with the followingoligonucleotides:

oligo-47 (SEQ ID NO. 68) 5′-ggc cgc aag ctt act gct gga tcc tta att aatcga tag tga tct cga ggc-3′ oligo-48 (SEQ ID NO. 69) 5′-ggc cgc ctc gagatc act atc gat taa tta agg atc cag cag taa gct tgc-3′

The resulting plasmid, pGP1b, contains a short polylinker region flankedby NotI sites. This facilitates the construction of large inserts thatcan be excised by NotI digestion.

3. pGPe

The following oligonucleotides:

oligo-44 (SEQ ID NO. 70) 5′-ctc cag gat cca gat atc agt acc tga aac agggct tgc-3′ oligo-45 (SEQ ID NO. 71) 5′-ctc gag cat gca cag gac ctg gagcac aca cag cct tcc -3′were used to amplify the immunoglobulin heavy chain 3′ enhancer (S.Petterson, et al., Nature 344: 165-168 (1990)) from rat liver DNA by thepolymerase chain reaction technique.

The amplified product was digested with BamHI and SphI and cloned intoBamHI/SphI digested pNNO3 (pNNO3 is a pUC derived plasmid that containsa polylinker with the following restriction sites, listed in order:NotI, BamHI, NcoI, ClaI, EcoRV, XbaI, SacI, XhoI, SphI, PstI, BglII,EcoRI, SmaI, KpnI, HindIII, and NotI). The resulting plasmid, pRE3, wasdigested with BamHI and HindIII, and the insert containing the rat Igheavy chain 3′ enhancer cloned into BamHI/HindIII digested pGP1b. Theresulting plasmid, pGPe (FIG. 22 and Table 1), contains several uniquerestriction sites into which sequences can be cloned and subsequentlyexcised together with the 3′ enhancer by NotI digestion.

TABLE 1 Sequence of vector pGPe.AATTAGCggccgcctcgagatcactatcgattaattaaggatccagatatcagtacctgaaacagggctgctcacaacatctctctctctgtctctctgtctctgtgtgtgtgtctctctctgtctctgtctctctctgtctctctgtctctgtgtgtgtctctctctgtctctctctctgtctctctgtctctctgtctgtctctgtctctgtctctgtctctctctctctctctctctctctctctctctctctcacacacacacacacacacacacacacacctgccgagtgactcactctgtgcagggttggccctcggggcacatgcaaatggatgtttgttccatgcagaaaaacatgtttctcattctctgagccaaaaatagcatcaatgattcccccaccctgcagctgcaggttcaccccacctggccaggttgaccagctttggggatggggctgggggttccatgacccctaacggtgacattgaattcagtgttttcccatttatcgacactgctggaatctgaccctaggagggaatgacaggagataggcaaggtccaaacaccccagggaagtgggagagacaggaaggctgtgtgtgctccaggtcctgtgcatgctgcagatctgaattcccgggtaccaagcttgcGGCCGCAGTATGCAAAAAAAAGCCCGCTCATTAGGCGGGCTCTTGGCAGAACATATCCATCGCGTCCGCCATCTCCAGCAGCCGCACGCGGCGCATCTCGGGCAGCGTTGGGTCCTGGCCACGGGTGCGCATGATCGTGCTCCTGTCGTTGAGGACCCGGCTAGGCTGGCGGGGTTGCCTTACTGGTTAGCAGAATGAATCACCGATACGCGAGCGAACGTGAAGCGACTGCTGCTGCAAAACGTCTGCGACCTGAGCAACAACATGAATGGTCTTCGGTTTCCGTGTTTCGTAAAGTCTGGAAACGCGGAAGTCAGCGCCCTGCACCATTATGTTCCGGATCTGCATCGCAGGATGCTGCTGGCTACCCTGTGGAACACCTACATCTGTATTAACGAAGCGCTGGCATTGACCCTGAGTGATTTTTCTCTGGTCCCGCCGCATCCATACCGCCAGTTGTTTACCCTCACAACGTTCCAGTAACCGGGCATGTTCATCATCAGTAACCCGTATCGTCACGATCCTCTCTCGTTTCATCGGTATCATTACCCCCATGAACAGAAATTCCCCCTTACACGGAGGCATCAAGTGACCAAACAGGAAAAAACCGCCCTTAACATGGCCCGCTTTATCAGAAGCCAGACATTAACGCTTCTGGAGAAACTCAACGAGCTGGACGCGGATGAACAGGCAGACATCTGTGAATCGCTTCACGACCACGCTGATGAGCTTTACCGCAGCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTCAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGCCAGGTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTGCAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTAGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCCACCGAGTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTGATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGA GGCCCTTTCGTCTTCAAGB. Construction of IgM Expressing Minilocus Transgene.pIGM11. Isolation of J-μ Constant Region Clones and Construction of pJM1

A human placental genomic DNA library cloned into the phage vectorλEMBL3/SP6/T7 (Clonetech Laboratories, Inc., Palo Alto, Calif.) wasscreened with the human heavy chain J region specific oligonucleotide:

oligo-1 (SEQ ID NO. 73) 5′-gga ctg tgt ccc tgt gtg atg ctt ttg atg tctggg gcc aag-3′

and the phage clone λ1.3 isolated. A 6 kb HindIII/KpnI fragment fromthis clone, containing all six J segments as well as D segment DHQ52 andthe heavy chain J-μ intronic enhancer, was isolated. The same librarywas screened with the human μ specific oligonucleotide:

oligo-2 (SEQ ID NO. 74) 5′-cac caa gtt gac ctg cct ggt cac aga cct gaccac cta tga-3′and the phage clone λ2.1 isolated. A 10.5 kb HindIII/XhoI fragment,containing the μ switch region and all of the μ constant region exons,was isolated from this clone. These two fragments were ligated togetherwith KpnI/XhoI digested pNNO3 to obtain the plasmid pJM1.

2. pJM2 A 4 kb XhoI fragment was isolated from phage clone λ2.1 thatcontains sequences immediately downstream of the sequences in pJM1,including the so called Σμ element involved in δ-associated deletion ofthe μ in certain IgD expressing B-cells (Yasui et al., Eur. J. Immunol.19: 1399 (1989), which is incorporated herein by reference). Thisfragment was treated with the Klenow fragment of DNA polymerase I andligated to XhoI cut, Klenow treated, pJM1. The resulting plasmid, pJM2(FIG. 23), had lost the internal XhoI site but retained the 3′ XhoI sitedue to incomplete reaction by the Klenow enzyme. pJM2 contains theentire human J region, the heavy chain J-μ intronic enhancer, the μswitch region and all of the μ constant region exons, as well as the two0.4 kb direct repeats, Σμ and Σμ, involved in δ-associated deletion ofthe μ gene.

3. Isolation of D Region Clones and Construction of pDH1

The following human D region specific oligonucleotide:

oligo-4 (SEQ ID NO. 75) 5′-tgg tat tac tat ggt tcg ggg agt tat tat aaccac agt gtc-3′was used to screen the human placenta genomic library for D regionclones. Phage clones λ4.1 and λ4.3 were isolated. A 5.5 kb XhoIfragment, that includes the D elements D_(K1), D_(N1), and D_(M2)(Ichihara et al., EMBO J. 7: 4141 (1988)), was isolated from phage cloneλ4.1. An adjacent upstream 5.2 kb XhoI fragment, that includes the Delements D_(LR1), D_(XP1), D_(XP′1), and D_(A1) was isolated from phageclone λ4.3. Each of these D region XhoI fragments were cloned into theSalI site of the plasmid vector pSP72 (Promega, Madison, Wis.) so as todestroy the XhoI site linking the two sequences. The upstream fragmentwas then excised with XhoI and SmaI, and the downstream fragment withEcoRV and XhoI. The resulting isolated fragments were ligated togetherwith SalI digested pSP72 to give the plasmid pDH1. pDH1 contains a 10.6kb. insert that includes at least 7 D segments and can be excised withXhoI (5′) and EcoRV (3′).4. pCOR1

The plasmid pJM2 was digested with Asp718 (an isoschizomer of KpnI) andthe overhang filled in with the Klenow fragment of DNA polymerase I. Theresulting DNA was then digested with ClaI and the insert isolated. Thisinsert was ligated to the XhoI/EcoRV insert of pDH1 and XhoI/ClaIdigested pGPe to generate pCOR1 (FIG. 24).

5. pVH251

A 10.3 kb genomic HindIII fragment containing the two human heavy chainvariable region segments V_(H) 251 and V_(H) 105 (Humphries et al.,Nature 331:446 (1988), which is incorporated herein by reference) wassubcloned into pSP72 to give the plasmid pVH251.

6. pIGM1

The plasmid pCOR1 was partially digested with XhoI and the isolatedXhoI/SalI insert of pVH251 cloned into the upstream XhoI site togenerate the plasmid pIGM1 (FIG. 25). pIGM1 contains 2 functional humanvariable region segments, at least 8 human D segments all 6 human J_(H)segments, the human J-μ enhancer, the human Σμ element, the human μswitch region, all of the human μ coding exons, and the human Σμelement, together with the rat heavy chain 3′ enhancer, such that all ofthese sequence elements can be isolated on a single fragment, away fromvector sequences, by digestion with NotI and microinjected into mouseembryo pronuclei to generate transgenic animals.

C. Construction of IgM and IgG Expressing Minilocus Transgene, pHC1

1. Isolation of γ Constant Region Clones

The following oligonucleotide, specific for human Ig g constant regiongenes:

oligo-29 (SEQ ID NO. 76) 5′-cag cag gtg cac acc caa tgc cca tga gcc cagaca ctg gac-3′

was used to screen the human genomic library. Phage clones 129.4 andλ29.5 were isolated. A 4 kb HindIII fragment of phage clone λ29.4,containing a γ switch region, was used to probe a human placenta genomicDNA library cloned into the phage vector lambda FIX™ II (Stratagene, LaJolla, Calif.). Phage clone λSg1.13 was isolated. To determine thesubclass of the different γ clones, dideoxy sequencing reactions werecarried out using subclones of each of the three phage clones astemplates and the following oligonucleotide as a primer:

oligo-67 5′-tga gcc cag aca ctg gac-3′ (SEQ ID NO. 77)

Phage clones λ29.5 and λSγ1.13 were both determined to be of the γ1subclass.

2. pγe1

A 7.8 kb HindIII fragment of phage clone λ29.5, containing the γ1 codingregion was cloned into pUC18. The resulting plasmid, pLT1, was digestedwith XhoI, Klenow treated, and religated to destroy the internal XhoIsite. The resulting clone, pLT1xk, was digested with HindIII and theinsert isolated and cloned into pSP72 to generate the plasmid clonepLT1xks. Digestion of pLT1xks at a polylinker XhoI site and a humansequence derived BamHI site generates a 7.6 kb fragment containing theγ1 constant region coding exons. This 7.6 kb XhoI/BamHI fragment wascloned together with an adjacent downstream 4.5 kb BamHI fragment fromphage clone λ29.5 into XhoI/BamHI digested pGPe to generate the plasmidclone pγe1. pγe1 contains all of the γ1 constant region coding exons,together with 5 kb of downstream sequences, linked to the rat heavychain 3′ enhancer.

3. pγe2

A 5.3 kb HindIII fragment containing the γ1 switch region and the firstexon of the pre-switch sterile transcript (P. Sideras et al. (1989)International Immunol. 1, 631) was isolated from phage clone λSγ1.13 andcloned into pSP72 with the polylinker XhoI site adjacent to the 5′ endof the insert, to generate the plasmid clone pSγ1s. The XhoI/SalI insertof pSγ1s was cloned into XhoI digested pγe1 to generate the plasmidclone pγe2 (FIG. 26). pγe2 contains all of the γ1 constant region codingexons, and the upstream switch region and sterile transcript exons,together with 5 kb of downstream sequences, linked to the rat heavychain 3′ enhancer. This clone contains a unique XhoI site at the 5′ endof the insert. The entire insert, together with the XhoI site and the 3′rat enhancer can be excised from vector sequences by digestion withNotI.

4. pHC1

The plasmid pIGM1 was digested with XhoI and the 43 kb insert isolatedand cloned into XhoI digested pge2 to generate the plasmid pHC1 (FIG.25). pHC1 contains 2 functional human variable region segments, at least8 human D segments all 6 human J_(H) segments, the human J-μ enhancer,the human Σμ element, the human μ switch region, all of the human μcoding exons, the human Σμ element, and the human γ1 constant region,including the associated switch region and sterile transcript associatedexons, together with the rat heavy chain 3′ enhancer, such that all ofthese sequence elements can be isolated on a single fragment, away fromvector sequences, by digestion with NotI and microinjected into mouseembryo pronuclei to generate transgenic animals.

D. Construction of IgM and IgG Expressing Minilocus Transgene, pHC2

1. Isolation of Human Heavy Chain V Region Gene VH49.8

The human placental genomic DNA library lambda, FIX™ II, Stratagene, LaJolla, Calif.) was screened with the following human VH1 family specificoligonucleotide:

oligo-49 (SEQ ID NO. 78) 5′-gtt aaa gag gat ttt att cac ccc tgt gtc ctctcc aca ggt gtc-3′

Phage clone λ49.8 was isolated and a 6.1 kb XbaI fragment containing thevariable segment VH49.8 subcloned into pNNO3 (such that the polylinkerClaI site is downstream of VH49.8 and the polylinker XhoI site isupstream) to generate the plasmid pVH49.8. An 800 bp region of thisinsert was sequenced, and VH49.8 found to have an open reading frame andintact splicing and recombination signals, thus indicating that the geneis functional (Table 2).

TABLE 2 Sequence of human V_(H)I family gene V_(H )49.8(SEQ. ID Nos. 79 and 80)

2. pV2

A 4 kb XbaI genomic fragment containing the human V_(H) IV family geneV_(H) 4-21 (Sanz et al., EMBO J., 8: 3741 (1989)), subcloned into theplasmid pUC12, was excised with SmaI and HindIII, and treated with theKlenow fragment of polymerase I. The blunt ended fragment-was thencloned into ClaI digested, Klenow treated, pVH49.8. The resultingplasmid, pV2, contains the human heavy chain gene VH49.8 linked upstreamof VH4-21 in the same orientation, with a unique SalI site at the 3′ endof the insert and a unique XhoI site at the 5′ end.

3. pSγ1-5′

A 0.7 kb XbaI/HindIII fragment (representing sequences immediatelyupstream of, and adjacent to, the 5.3 kb γ1 switch region containingfragment in the plasmid pγe2) together with the neighboring upstream 3.1kb XbaI fragment were isolated from the phage clone λSg1.13 and clonedinto HindIII/XbaI digested pUC18 vector. The resulting plasmid, pSγ1-5′,contains a 3.8 kb insert representing sequences upstream of theinitiation site of the sterile transcript found in B-cells prior toswitching to the γ1 isotype (P. Sideras et al., International Immunol.1: 631 (1989)). Because the transcript is implicated in the initiationof isotype switching, and upstream cis-acting sequences are oftenimportant for transcription regulation, these sequences are included intransgene constructs to promote correct expression of the steriletranscript and the associated switch recombination.

4. pVGE1

The pSγ1-5′ insert was excised with SmaI and HindIII, treated withKlenow enzyme, and ligated with the following oligonucleotide linker:

5′-ccg gtc gac cgg-3′ (SEQ ID NO. 81)The ligation product was digested with SalI and ligated to SalI digestedpV2. The resulting plasmid, pVP, contains 3.8 kb of γ1 switch 5′flanking sequences linked downstream of the two human variable genesegments VH49.8 and VH4-21 (see Table 2). The pVP insert is isolated bypartial digestion with SalI and complete digestion with XhoI, followedby purification of the 15 kb fragment on an agarose gel. The insert isthen cloned into the XhoI site of pγe2 to generate the plasmid clonepVGE1 (FIG. 27). pVGE1 contains two human heavy chain variable genesegments upstream of the human γ1 constant gene and associated switchregion. A unique SalI site between the variable and constant regions canbe used to clone in D, J, and μ gene segments. The rat heavy chain 3′enhancer is linked to the 3′ end of the γ1 gene and the entire insert isflanked by NotI sites.5. pHC2

The plasmid clone pVGE1 is digested with SalI and the XhoI insert ofpIGM1 is cloned into it. The resulting clone, pHC2 (FIG. 25), contains 4functional human variable region segments, at least 8 human D segmentsall 6 human J_(H) segments, the human J-m enhancer, the human Σμelement, the human μ switch region, all of the human μ coding exons, thehuman Σμ element, and the human γ1 constant region, including theassociated switch region and sterile transcript associated exons,together with 4 kb flanking sequences upstream of the sterile transcriptinitiation site. These human sequences are linked to the rat heavy chain3′ enhancer, such that all of the sequence elements can be isolated on asingle fragment, away from vector sequences, by digestion with NotI andmicroinjected into mouse embryo pronuclei to generate transgenicanimals. A unique XhoI site at the 5′ end of the insert can be used toclone in additional human variable gene segments to further expand therecombinational diversity of this heavy chain minilocus.

E. Transgenic Mice

The NotI inserts of plasmids pIGM1 and pHC1 were isolated from vectorsequences by agarose gel electrophoresis. The purified inserts weremicroinjected into the pronuclei of fertilized (C57BL/6xCBA)F2 mouseembryos and transferred the surviving embryos into pseudopregnantfemales as described by Hogan et al. (B. Hogan, F. Costantini, and E.Lacy, Methods of Manipulating the Mouse Embryo, 1986, Cold Spring HarborLaboratory, New York). Mice that developed from injected embryos wereanalyzed for the presence of transgene sequences by Southern blotanalysis of tail DNA. Transgene copy number was estimated by bandintensity relative to control standards containing known quantities ofcloned DNA. At 3 to 8 weeks of age, serum was isolated from theseanimals and assayed for the presence of transgene encoded human IgM andIgG1 by ELISA as described by Harlow and Lane (E. Harlow and D. Lane.Antibodies: A Laboratory Manual, 1988, Cold Spring Harbor Laboratory,New York). Microtiter plate wells were coated with mouse monoclonalantibodies specific for human IgM (clone AF6, #0285, AMAC, Inc.Westbrook, Me.) and human IgG1 (clone JL512, #0280, AMAC, Inc.Westbrook, Me.). Serum samples were serially diluted into the wells andthe presence of specific immunoglobulins detected with affinity isolatedalkaline phosphatase conjugated goat anti-human Ig (polyvalent) that hadbeen pre-adsorbed to minimize cross-reactivity with mouseimmunoglobulins. Table 3 and FIG. 28 show the results of an ELISA assayfor the presence of human IgM and IgG1 in the serum of two animals thatdeveloped from embryos injected with the transgene insert of plasmidpHC1. All of the control non-transgenic mice tested negative forexpression of human IgM and IgG1 by this assay. Mice from two linescontaining the pIGM1 NotI insert (lines #6 and 15) express human IgM butnot human IgG1. We tested mice from 6 lines that contain the pHC1 insertand found that 4 of the lines (lines #26, 38, 57 and 122) express bothhuman IgM and human IgG1, while mice from two of the lines (lines #19and 21) do not express detectable levels of human immunoglobulins. ThepHC1 transgenic mice that did not express human immunoglobulins wereso-called G_(o) mice that developed directly from microinjected embryosand may have been mosaic for the presence of the transgene. Southernblot analysis indicates that many of these mice contain one or fewercopies of the transgene per cell. The detection of human IgM in theserum of pIGM1 transgenics, and human IgM and IgG1 in pHC1 transgenics,provides evidence that the transgene sequences function correctly indirecting VDJ joining, transcription, and isotype switching. One of theanimals (#18) was negative for the transgene by Southern blot analysis,and showed no detectable levels of human IgM or IgG1. The second animal(#38) contained approximately 5 copies of the transgene, as assayed bySouthern blotting, and showed detectable levels of both human IgM andIgG1. The results of ELISA assays for 11 animals that developed fromtransgene injected embryos is summarized in the table below (Table 3).

TABLE 3 Detection of human IgM and IgG1 in the serum of transgenicanimals by ELISA assay approximate injected transgene animal # transgenecopies per cell human IgM human IgG1 6 pIGM1 1 ++ − 7 pIGM1 0 − − 9pIGM1 0 − − 10 pIGM1 0 − − 12 pIGM1 0 − − 15 pIGM1 10 ++ − 18 pHC1 0 − −19 pHC1 1 − − 21 pHC1 <1 − − 26 pHC1 2 ++ + 38 pHC1 5 ++ +

Table 3 shows a correlation between the presence of integrated transgeneDNA and the presence of transgene encoded immunoglobulins in the serum.Two of the animals that were found to contain the pHC1 transgene did notexpress detectable levels of human immunoglobulins. These were both lowcopy animals and may not have contained complete copies of thetransgenes, or the animals may have been genetic mosaics (indicated bythe <1 copy per cell estimated for animal #21), and the transgenecontaining cells may not have populated the hematopoietic lineage.Alternatively, the transgenes may have integrated into genomic locationsthat are not conducive to their expression. The detection of human IgMin the serum of pIGM1 transgenics, and human IgM and IgG1 in pHC1transgenics, indicates that the transgene sequences function correctlyin directing VDJ joining, transcription, and isotype switching.

F. cDNA Clones

To assess the functionality of the pHC1 transgene in VDJ joining andclass switching, as well the participation of the transgene encodedhuman B-cell receptor in B-cell development and allelic exclusion, thestructure of immunoglobulin cDNA clones derived from transgenic mousespleen mRNA were examined. The overall diversity of the transgeneencoded heavy chains, focusing on D and J segment usage, N regionaddition, CDR3 length distribution, and the frequency of jointsresulting in functional mRNA molecules was examined. Transcriptsencoding IgM and IgG incorporating VH105 and VH251 were examined.

Polyadenylated RNA was isolated from an eleven week old male secondgeneration line-57 pHC1 transgenic mouse. This RNA was used tosynthesize oligo-dT primed single stranded cDNA. The resulting cDNA wasthen used as template for four individual PCR amplifications using thefollowing four synthetic oligonucleotides as primers: VH251 specificoligo-149, cta gct cga gtc caa gga gtc tgt gcc gag gtg cag ctg (g,a,t,c)(SEQ ID NO.: 82); VH105 specific o-150, gtt gct cga gtg aaa ggt gtc cagtgt gag gtg cag ctg (g,a,t,c) (SEQ ID NO.: 83); human gammal specificoligo-151, ggc gct cga gtt cca cga cac cgt cac cgg ttc (SEQ ID NO.: 84);and human mu specific oligo-152, cct gct cga ggc agc caa cgg cca cgc tgctcg. (SEQ ID NO.: 85) Reaction 1 used primers 0-149 and o-151 to amplifyVH251-gammal transcripts, reaction 2 used o-149 and o-152 to amplifyVH251-mu transcripts, reaction 3 used o-150 and o-151 to amplifyVH105-gammal transcripts, and reaction 4 used o-150 and o-152 to amplifyVH105-mu transcripts. The resulting 0.5 kb PCR products were isolatedfrom an agarose gel; the μ transcript products were more abundant thanthe γ transcript products, consistent with the corresponding ELISA data(FIG. 34). The PCR products were digested with XhoI and cloned into theplasmid pNNO3. Double-stranded plasmid DNA was isolated from miniprepsof nine clones from each of the four PCR amplifications and dideoxysequencing reactions were performed. Two of the clones turned out to bedeletions containing no D or J segments. These could not have beenderived from normal RNA splicing products and are likely to haveoriginated from deletions introduced during PCR amplification. One ofthe DNA samples turned out to be a mixture of two individual clones, andthree additional clones did not produce readable DNA sequence(presumably because the DNA samples were not clean enough). The DNAsequences of the VDJ joints from the remaining 30 clones are compiled inTable 4. Each of the sequences are unique, indicating that no singlepathway of gene rearrangement, or single clone of transgene expressingB-cells is dominant. The fact that-no two sequences are alike is also anindication of the large diversity of immunoglobulins that can beexpressed from a compact minilocus containing only 2 V segments, 10 Dsegments, and 6 J segments. Both of the V segments, all six of the Jsegments, and 7 of the 10 D segments that are included in the transgeneare used in VDJ joints. In addition, both constant region genes (mu andgammal) are incorporated into transcripts. The VH105 primer turned outnot to be specific for VH105 in the reactions performed. Therefore manyof the clones from reactions 3 and 4 contained VH251 transcripts.Additionally, clones isolated from ligated reaction 3 PCR product turnedout to encode IgM rather than IgG; however this may reflectcontamination with PCR product from reaction 4 as the DNA was isolatedon the same gel. An analogous experiment, in which immunoglobulin heavychain sequences were amplified from adult human peripheral bloodlymphocytes (PBL), and the DNA sequence of the VDJ joints determined,was recently reported by Yamada et al. (J. Exp. Med. 173: 395-407(1991), which is incorporated herein by reference). We compared the datafrom human PBL with our data from the pHC1 transgenic mouse.

TABLE 4 V n-D-n  1 VH251 DHQ52 J3 γ1 TACTGTGCGAGA CGGCTAAACTGGGGTTGAT  2VH251 DN1 J4 γ1 TACTGTGCGAGA CACCGTATAGCAGCAGCTGG  3 VH251 DN1 J6 γ1TACTGTGCGAGA T  4 VH251 D? J6 γ1 TACTGTGCGAGA CATTACGATATTTTGACTGGTC  5VH251 DXP′1 J4 γ1 TACTGTGCGAGA CGGAGGTACTATGGTTCGGGGAGTTATTATAACGT  6VH251 D? J3 γ1 TACTGTGCGAGA CGGGGGGTGTCTGAT  7 VH251 DHQ52 J3 μTACTGTGCGAGA GCAACTGGC  8 VH251 DHQ52 J6 μ TACTGTGCGACATCGGCTAACTGGGGATC  9 VH251 — J1 μ TACTGTGCGAGA 10 VH251 DLR2 J4 μTACTGTGCGAGA CACGTAGCTAACTCT 11 VH251 DXP′1 J4 μ TACTGTGCGAGACAAATTACTATGGTTCGGGGAGTTCC 12 VH251 D? J1 μ TACTGTGCGAGA C 13 VH251DHQ52 J6 μ TACTGTGCGAGA CAAACTGGGG 14 VH251 DXP′1 J6 γ1 TACTGTGCGAGACATTACTATGGTTCGGGGAGTTATG 15 VH251 DXP′1 J4 μ TACTGTGCGAGA CAGGGAG 16VH105 DXP′1 J5 μ TACTGTGCGAGA TTCTGGGAG 17 VH251 DXP′1 J4 γ1TACTGTGCGAGA CGGAGGTACTATGGTTCGGGGAGTTATTATAACGT 18 VH251 DHQ52 J4 γ1TACTGTGCGAGA CAAACCTGGGGAGGA 19 VH251 DK1 J6 γ1 TACTCTGCGAGAGGATATAGTGGCTACGATA 20 VH251 DHQ52 J4 γ TACTGTGCGAGA CAAACTGGGGAGG 21VH251 DK1 J2 γ1 TACTGRGCGAGA TATAGTGGCTACGATTAC 22 VH251 DIR2 J6 γ1TACTGRGCGAGA GCATCCCTCCCCTCCTTTG 23 VH251 DIR2 J4 μ TACTGTGCGAGACGGGGTGGGG 24 VH105 D? J6 μ TACTGTGTG CCGGTCGAAACT 25 VH105 DXP′1 J4 μTACTGTGCGAGA GATATTTTGACTGGTTAACG 26 VH251 DN1 J3 μ TACTGTGCGAGACATGGTATAGCAGCAGCTGGTAC 27 VH105 DHQ52 J3 μ TACTGTGTGAGA TCAACTGGGGTTG28 VH251 DN1 J4 μ TACTGTGCG GAAATAGCAGCAGCTGCC 29 VH105 DN1 J4 μTACTGTGTG TGTATAGCAGCAGCTGGTAAAGGAAACGG 30 VH251 DHQ52 J4 μ TACTGTGCGAGACAAAACTGGGG                                                              i C  1VH251 DHQ52 J3 γ1                 GCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAGCCTCCAC-CAAG  2 VH251 DN1 J4 γ1                   CTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCAC-CAAG  3 VH251 DN1 J6 γ1ATTACTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCTCCAC-CAAG  4 VH251 D? J6 γ1    CTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCTCCAC-CAAG  5 VH251 DXP′1 J4 γ1                    CTTTGACTACTGGGGCCAGGGAACCTGGTCACCGTCTCCTCAGCCTCCAC-CAAG  6 VH251 D? J3 γ1                  GCTTTTGATATCTGGGCCAAGGGACAATGGTCACCGTCTCTTCAGCCTCCAC-CAAG  7 VH251 DHQ52 J3 μ                 GCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAGGGAGTG-CATCC  8 VH251 DHQ52 J6 μ    CTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGGGAGTG-CATCC  9 VH251 — J1 μ                 TACTTCCAGCACTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGGGAGT-GCGTCC 10 VH251 DLR2 J4 μ                    TTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGGAGTG-CATCC 11 VH251 DXP′1 J4 μ                   CTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGGAGTG-CATCC 12 VH251 D? J1 μ                AATACTTCCAGCACTGGGGCCAGGGCACCTGGTCACCGTCTCCTCAGGGAGTG-CATCC 13 VH251 DHQ52 J6 μ   ACTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGGGAGTG-CATCC 14 VH251 DXP′1 J6 γ1   ACTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGGGAGTG-CATCC 15 VH251 DXP′1 J4 μ                             TGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCAC-CAAG 16 VH105 DXP′1 J5 μ                ACTGGTTCGACCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGGAGTG-CATCC 17 CH251 DXP′1 J4 γ1                   CTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCAC-CAAG 18 VH251 DHQ52 J4 γ1                       GACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCAC-CAAG 19 VH251 DK1 J6 γ1      ACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCTCCAC-CAAG 20 VH251 DHQ52 J4 μ               ACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGGAGTG-CATCC 21 VH251 DK1 J2 γ1          CTACTGGTACTTCGATCTCTGGGGCCGTGGCACCCTGGTCACCGTCTCCTCAGCCTCCAC-CAAG 22 VH251 DIR2 J6 γ1         ACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCCTCCAC-CAAG 23 VH251 DIR2 J4 μ                     TTTGACTACTGGGGCCAGGGAACCTGGTCACCGTCTCCTCAGGGAGTG-CATCC 24 VH105 D? J6 μ TTACTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGGGAGTG-CATCC 25 VH105 DXP′1 J4 μ                      TGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGGAGTG-CATCC 26 VH251 DN1 J3 μ                TGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCCTCAGGGAGTG-CATCC 27 VH105 DHQ52 J3 μ               ATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCCTCAGGGAGTG-CATCC 28 VH251 DN1 J4 μ                CTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGGAGTG-CATCC 29 VH105 DN1 J4 μ                        CTACTGGGGCCAGGGGAACCCTGGTCACCGTCTCCTCAGGGAGTG-CATCC 30 VH251 DHQ52 J4 μ                      TGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGGAGTG-CATCCG. J Segment Choice

Table 5 compared the distribution of J segments incorporated into pHC1transgene encoded transcripts to J segments found in adult human PBLimmunoglobulin transcripts. The distribution profiles are very similar,J4 is the dominant segment in both systems, followed by J6. J2 is theleast common segment in human PBL and the transgenic animal.

TABLE 5 J. Segment Choice Percent Usage (±3%) J. Segment HC1 transgenicHuman PBL J1 7 1 J2 3 <1 J3 17 9 J4 44 53 J5 3 15 J6 26 22 100% 100%H. D Segment Choice

49% (40 of 82) of the clones analyzed by Yamada et al. incorporated Dsegments that are included in the pHC1 transgene. An additional 11clones contained sequences that were not assigned by the authors to anyof the known D segments. Two of these 11 unassigned clones appear to bederived from an inversion of the DIR2 segments which is included in thepHC1 construct. This mechanism, which was predicted by Ichihara et al.(EMBO J. 7: 4141 (1988)) and observed by Sanz (J. Immunol. 147:1720-1729 (1991)), was not considered by Yamada et al. (J. Exp. Med.173: 395-407 (1991)). Table 5 is a comparison of the D segmentdistribution for the pHC1 transgenic mouse and that observed for humanPBL transcripts by Yamada et al. The data of Yamada et al. wasrecompiled to include DIR2 use, and to exclude D segments that are notin the pHC1 transgene. Table 6 demonstrates that the distribution of Dsegment incorporation is very similar in the transgenic mouse and inhuman PBL. The two dominant human D segments, DXP′1 and DN1, are alsofound with high frequency in the transgenic mouse. The most dramaticdissimilarity between the two distributions is the high frequency ofDHQ52 in the transgenic mouse as compared to the human. The highfrequency of DHQ52 is reminiscent of the D segment distribution in thehuman fetal liver. Sanz has observed that 14% of the heavy chaintranscripts contained DHQ52 sequences. If D segments not found in pHC1are excluded from the analysis, 31% of the fetal transcripts analyzed bySanz contain DHQ52. This is comparable to the 27% that we observe in thepHC1 transgenic mouse.

TABLE 6 D Segment Choice Percent Usage (±3%) D. Segment HC1 transgenicHuman PBL DLR1 <1 <1 DXP1 3 6 DXP′1 25 19 DA1 <1 12 DK1 7 12 DN1 12 22DIR2 7 4 DM2 <1 2 DLR2 3 4 DHQ52 26 2 ? 17 17 100% 100%I. Functionality of VDJ Joints

Table 7 shows the predicted amino acid sequences of the VDJ regions from30 clones that were analyzed from the pHC1 transgenic. The translatedsequences indicate that 23 of the 30 VDJ joints (77%) are in-frame withrespect to the variable and J segments.

TABLE 7 Functionality of VD-J Joints FR 3 CDR3 FR4  1 VH251 DHQ52 J3 γ1YCAR RLTGVDAFDI WGQGTMVTMSSASTK  2 VH251 DN1 J4 γ1 YCAR HRIAAAGFDYWGQGTLVTVSSASTK  3 VH251 D? J6 γ1 YCAR YYYYYYGMDV WGQGTTVTVSSASTK  4VH251 DXP′1 J6 γ1 YCAR HYDILTGPTTTTVWTSGAKGPRSPSPQPPP  5 VH251 DXP′1 J4γ1 YCAR RRYYGSGSYYNVFDY WGQGTLVTVSSADTK  6 VH251 D? J3 γ1 YCAR RGVSDAFDIWGQGTMVTVSSADTK  7 VH251 DHQ52 J3 μ YCAR ATGAFDI WGQGTMVTVSSGSAS  8VH251 DHQ52 J6 μ YCAR SANWGSYYYYGMDV WGQGTTVTVSSGSAS  9 VH251 — J1 μYCAR YFQH WGQGTLVTVSSGSAS 10 VH251 DLR2 J4 μ YCAR HVANSFDYWGQGTLVTVSSGSAS 11 VH251 DXP′1 J4 μ YCAR QITMVRGVPFDY WGQGTLVTVSSGSAS 12VH251 D? J1 μ YCAR QYFQH WGQGTLVTVSSGSAS 13 VH251 DHQ52 J6 μ YCARQTGDYYYYGMDV WGQGTTVTVSSGSAS 14 VH251 DXP′1 J6 μ YCAR HYYGSGSYDYYYYGMDVWGQGTTVTVSSGSAS 15 VH251 DXP′1 J4 γ1 YCAR QGVGPGNPGHRLLSLHQ 16 VH105DXP′1 J5 μ YCVR FWETGSTPGAREPWSPSPQGVH 17 VH251 DXP′1 J4 γ1 YCARRRYYGSGSYYNVFDY WGQGTLVTVSSGSTK 18 VH251 DHQ52 J4 γ1 YCAR QTWGGDYWGQGTLVTVSSGSTK 19 VH251 DK1 J6 γ1 YCAR GYSGYDNYYYGIHV WGQGTTVTVSSGSTK20 VH251 DHQ52 J4 μ YCAR QTGEDYFDY WGQGTLVTVSSGSAS 21 VH251 DK1 J2 γ1YCAR YSGYDYLLVLRSLGPWHPGHCLLSLHR 22 VH251 DIR2 J6 γ1 YCAR ASLPSFDYYGMDVWGQGTTVTVSSGSTK 23 VH251 DIR2 J4 μ YCAR RGGGLTTGAREPWSPSPQGVH 24 VH105D? J6 μ YCVP VETLLLLLRYGRLGPRDHGHRLLRECI 25 VH105 DXP1 J4 μ YCVRDILTGZRDY WGQGTLVTVSSGSAS 26 VH251 DM1 J3 μ YCAR HGIAAAGTAFDIWGQGTMVTVSSGSAS 27 VH105 DHQ52 J3 μ YCVR STGVDAFDI WGQGTMVTVSSGSAS 28VH251 DN1 J4 μ YCAE IAAAALLZLLGPGNPGHRLLRECI 29 VH105 DN1 J4 μ YCVCIAAAGKGNGY WGQGTLVTVSSGSAS 30 VH251 DHQ52 J4 μ YCAR QNWGDYWGQGTLVTVSSGSASJ. CDR3 Length Distribution

Table 8 compared the length of the CDR3 peptides from transcripts within-frame VDJ joints in the pHC1 transgenic mouse to those in human PBL.Again the human PBL data comes from Yamada et al. The profiles aresimilar with the transgenic profile skewed slightly toward smaller CDR3peptides than observed from human PBL. The average length of CDR3 in thetransgenic mouse is 10.3 amino acids. This is substantially the same asthe average size reported for authentic human CDR3 peptides by Sanz (J.Immunol. 147: 1720-1729 (1991)).

TABLE 8 CDR3 Length Distribution Percent Occurrence (±3%) #amino acidsin CDR3 HC1 transgenic Human PBL 3-8 26 14  9-12 48 41 13-18 26 37 19-23<1 7 >23 <1 1 100% 100%

Example 13 Rearranged Heavy Chain Transgenes

A. Isolation of Rearranged Human Heavy Chain VDJ Segments.

Two human leukocyte genomic DNA libraries cloned into the phage vectorλEMBL3/SP6/T7 (Clonetech Laboratories, Inc., Palo Alto, Calif.) arescreened with a 1 kb PacI/HindIII fragment of λ1.3 containing the humanheavy chain J-μ intronic enhancer. Positive clones are tested forhybridization with a mixture of the following V_(H) specificoligonucleotides:

oligo-7 (SEQ ID NO. 146) 5′-tca gtg aag gtt tcc tgc aag gca tct gga tacacc ttc acc-3′ oligo-8 (SEQ ID NO. 147) 5′-tcc ctg aga ctc tcc tgt gcagcc tct gga ttc acc ttc agt-3′

Clones that hybridized with both V and J-p probes are isolated and theDNA sequence of the rearranged VDJ segment determined.

B. Construction of Rearranged Human Heavy Chain Transgenes

Fragments containing functional VJ segments (open reading frame andsplice signals) are subcloned into the plasmid vector pSP72 such thatthe plasmid derived XhoI site is adjacent to the 5′ end of the insertsequence. A subclone containing a functional VDJ segment is digestedwith XhoI and PacI (PacI, a rare-cutting enzyme, recognizes a site nearthe J-m intronic enhancer), and the insert cloned into XhoI/PacIdigested pHC2 to generate a transgene construct with a functional VDJsegment, the J-μ intronic enhancer, the μ switch element, the μ constantregion coding exons, and the γ1 constant region, including the steriletranscript associated sequences, the γ1 switch, and the coding exons.This transgene construct is excised with NotI and microinjected into thepronuclei of mouse embryos to generate transgenic animals as describedabove.

Example 14 Light Chain Transgenes

A. Construction of Plasmid Vectors

1. Plasmid Vector pGP1c

Plasmid vector pGP1a is digested with NotI and the followingoligonucleotides ligated in:

oligo-81 (SEQ ID NO. 148) 5′-ggc cgc atc ccg ggt ctc gag gtc gac aag ctttcg agg atc cgc-3′ oligo-82 (SEQ ID NO. 149) 5′-ggc cgc gga tcc tcg aaagct tgt cga cct cga gac ccg gga tgc-3′The resulting plasmid, pGP1c, contains a polylinker with XmaI, XhoI,SalI, HindIII, and BamHI restriction sites flanked by NotI sites.2. Plasmid Vector pGP1d

Plasmid vector pGP1a is digested with NotI and the followingoligonucleotides ligated in:

oligo-87 (SEQ ID NO. 150) 5′-ggc cgc tgt cga caa gct tat cga tgg atc ctcgag tgc-3′ oligo-88 (SEQ ID NO. 151) 5′-ggc cgc act cga gga tcc atc gataag ctt gtc gac agc-3′The resulting plasmid, pGP1d, contains a polylinker with SalI, HindIII,ClaI, BamHI, and XhoI restriction sites flanked by NotI sites.B. Isolation of Jκ and Cκ Clones

A human placental genomic DNA library cloned into the phage vectorλEMBL3/SP6/T7 (Clonetech Laboratories, Inc., Palo Alto, Calif.) wasscreened with the human kappa light chain J region specificoligonucleotide:

oligo-36 (SEQ ID NO. 152) 5′-cac ctt cgg cca agg gac acg act gga gat taaacg taa gca-3′and the phage clones 136.2 and 136.5 isolated. A 7.4 kb XhoI fragmentthat includes the Jκ1 segment was isolated from 136.2 and subcloned intothe plasmid pNNO3 to generate the plasmid clone p36.2. A neighboring 13kb XhoI fragment that includes Jk segments 2 through 5 together with theCκ gene segment was isolated from phage clone 136.5 and subcloned intothe plasmid pNNO3 to generate the plasmid clone p36.5. Together thesetwo clones span the region beginning 7.2 kb upstream of Jκ1 and ending 9kb downstream of Cκ.C. Construction of Rearranged Light Chain Transgenes

1. pCK1, a Cκ Vector for Expressing Rearranged Variable Segments

The 13 kb XhoI insert of plasmid clone p36.5 containing the Cκ gene,together with 9 kb of downstream sequences, is cloned into the SalI siteof plasmid vector pGP1c with the 5′ end of the insert adjacent to theplasmid XhoI site. The resulting clone, pCK1 can accept cloned fragmentscontaining rearranged VJκ segments into the unique 5′ XhoI site. Thetransgene can then be excised with NotI and purified from vectorsequences by gel electrophoresis. The resulting transgene construct willcontain the human J-Cκ intronic enhancer and may contain the human 3′ κenhancer.

2. pCK2, a Cκ Vector with Heavy Chain Enhancers for ExpressingRearranged Variable Segments

A 0.9 kb XbaI fragment of mouse genomic DNA containing the mouse heavychain J-μ intronic enhancer (J. Banerji et al., Cell 33: 729-740 (1983))was subcloned into pUC18 to generate the plasmid pJH22.1. This plasmidwas linearized with SphI and the ends filled in with Klenow enzyme. TheKlenow treated DNA was then digested with HindIII and a 1.4 kbMluI/HindIII fragment of phage clone λ1.3 (previous example), containingthe human heavy chain J-μ intronic enhancer (Hayday et al., Nature 307:334-340 (1984)), to it. The resulting plasmid, pMHE1, consists of themouse and human heavy chain J-μ intronic enhancers ligated together intopUC18 such that they are excised on a single BamHI/HindIII fragment.This 2.3 kb fragment is isolated and cloned into pGP1c to generatepMHE2. pMHE2 is digested with SalI and the 13 kb XhoI insert of p36.5cloned in. The resulting plasmid, pCK2, is identical to pCK1, exceptthat the mouse and human heavy chain J-μ intronic enhancers are fused tothe 3′ end of the transgene insert. To modulate expression of the finaltransgene, analogous constructs can be generated with differentenhancers, i.e. the mouse or rat 3′ kappa or heavy chain enhancer (Meyerand Neuberger, EMBO J., 8: 1959-1964 (1989); Petterson et al., Nature,344: 165-168 (1990)).

3. Isolation of Rearranged Kappa Light Chain Variable Segments

Two human leukocyte genomic DNA libraries cloned into the phage vectorλEMBL3/SP6/T7 (Clonetech Laboratories, Inc., Palo Alto, Calif.) werescreened with the human kappa light chain J region containing 3.5 kbXhoI/SmaI fragment of p36.5. Positive clones were tested forhybridization with the following Vκ specific oligonucleotide:

oligo-65 (SEQ ID NO. 153) 5′-agg ttc agt ggc agt ggg tct ggg aca gac ttcact ctc acc atc agc-3′Clones that hybridized with both V and J probes are isolated and the DNAsequence of the rearranged VJκ segment determined. (SEQ ID NO.153)4. Generation of Transgenic Mice Containing Rearranged Human Light ChainConstructs.

Fragments containing functional VJ segments (open reading frame andsplice signals) are subcloned into the unique XhoI sites of vectors pCK1and pCK2 to generate rearranged kappa light chain transgenes. Thetransgene constructs are isolated from vector sequences by digestionwith NotI. Agarose gel purified insert is microinjected into mouseembryo pronuclei to generate transgenic animals. Animals expressinghuman kappa chain are bred with heavy chain minilocus containingtransgenic animals to generate mice expressing fully human antibodies.

Because not all VJκ combinations may be capable of forming stableheavy-light chain complexes with a broad spectrum of different heavychain VDJ combinations, several different light chain transgeneconstructs are generated, each using a different rearranged VJk clone,and transgenic mice that result from these constructs are bred withheavy chain minilocus transgene expressing mice. Peripheral blood,spleen, and lymph node lymphocytes are isolated from double transgenic(both heavy and light chain constructs) animals, stained withfluorescent antibodies specific for human and mouse heavy and lightchain immunoglobulins (Pharmingen, San Diego, Calif.) and analyzed byflow cytometry using a FACScan analyzer (Becton Dickinson, San Jose,Calif.). Rearranged light chain transgenes constructs that result in thehighest level of human heavy/light chain complexes on the surface of thehighest number of B cells, and do not adversely affect the immune cellcompartment (as assayed by flow cytometric analysis with B and T cellsubset specific antibodies), are selected for the generation of humanmonoclonal antibodies.

D. Construction of Unrearranged Light Chain Minilocus Transgenes

1. pJCK1, a Jκ, Cκ Containing Vector for Constructing MinilocusTransgenes

The 13 kb Cκ containing XhoI insert of p36.5 is treated with Klenowenzyme and cloned into HindIII digested, Klenow-treated, plasmid pGP1d.A plasmid clone is selected such that the 5′ end of the insert isadjacent to the vector derived ClaI site. The resulting plasmid,p36.5-1d, is digested with ClaI and Klenow-treated. The Jκ1 containing7.4 kb XhoI insert of p36.2 is then Klenow-treated and cloned into theClaI, Klenow-treated p36.5-1d. A clone is selected in which the p36.2insert is in the same orientation as the p36.5 insert. This clone, pJCK1(FIG. 34), contains the entire human Jκ region and Cκ, together with 7.2kb of upstream sequences and 9 kb of downstream sequences. The insertalso contains the human J-Cκ intronic enhancer and may contain a human3′ κ enhancer. The insert is flanked by a unique 3′ SalI site for thepurpose of cloning additional 3′ flanking sequences such as heavy chainor light chain enhancers. A unique XhoI site is located at the 5′ end ofthe insert for the purpose of cloning in unrearranged Vκ gene segments.The unique SalI and XhoI sites are in turn flanked by NotI sites thatare used to isolate the completed transgene construct away from vectorsequences.

2. Isolation of Unrearranged Vκ Gene Segments and Generation ofTransgenic Animals Expressing Human Ig Light Chain Protein

The Vκ specific oligonucleotide, oligo-65 (discussed above), is used toprobe a human placental genomic DNA library cloned into the phage vector1EMBL3/SP6/T7 (Clonetech Laboratories, Inc., Palo Alto, Calif.).Variable gene segments from the resulting clones are sequenced, andclones that appear functional are selected. Criteria for judgingfunctionality include: open reading frames, intact splice acceptor anddonor sequences, and intact recombination sequence. DNA fragmentscontaining selected variable gene segments are cloned into the uniqueXhoI site of plasmid pJCK1 to generate minilocus constructs. Theresulting clones are digested with NotI and the inserts isolated andinjected into mouse embryo pronuclei to generate transgenic animals. Thetransgenes of these animals will undergo V to J joining in developingB-cells. Animals expressing human kappa chain are bred with heavy chainminilocus containing transgenic animals to generate mice expressingfully human antibodies.

Example 15 Genomic Heavy Chain Human Ig Transgene

This Example describes the cloning of a human genomic heavy chainimmunoglobulin transgene which is then introduced into the murinegermline via microinjection into zygotes or integration in ES cells.

Nuclei are isolated from fresh human placental tissue as described byMarzluff, W. F., et al. (1985), Transcription and Translation: APractical Approach, B. D. Hammes and S. J. Higgins, eds., pp. 89-129,IRL Press, Oxford). The isolated nuclei (or PBS washed humanspermatocytes) are embedded in 0.5% low melting point agarose blocks andlysed with 1 mg/ml proteinase K in 500 mM EDTA, 1% SDS for nuclei, orwith 1 mg/ml proteinase K in 500 mM EDTA, 1% SDS, 10 mM DTT forspermatocytes at 50° C. for 18 hours. The proteinase K is inactivated byincubating the blocks in 40 μg/ml PMSF in TE for 30 minutes at 50° C.,and then washing extensively with TE. The DNA is then digested in theagarose with the restriction enzyme NotI as described by M. Finney inCurrent Protocols in Molecular Biology (F. Ausubel et al., eds. JohnWiley & Sons, Supp. 4, 1988, e.g., Section 2.5.1).

The NotI digested DNA is then fractionated by pulsed field gelelectrophoresis as described by Anand et al., Nuc. Acids Res. 17:3425-3433 (1989). Fractions enriched for the NotI fragment are assayedby Southern hybridization to detect one or more of the sequences encodedby this fragment. Such sequences include the heavy chain D segments, Jsegments, and γ1 constant regions together with representatives of all 6V_(H) families (although this fragment is identified as 670 kb fragmentfrom HeLa cells by Berman et al. (1988), supra., we have found it to bean 830 kb fragment from human placental and sperm DNA). Those fractionscontaining this NotI fragment are ligated into the NotI cloning site ofthe vector pYACNN as described (McCormick et al., Technique 2: 65-71(1990)). Plasmid pYACNN is prepared by digestion of pYACneo (Clontech)with EcoRI and ligation in the presence of the oligonucleotide 5′-AATTGC GGC CGC-3′ (25)

YAC clones containing the heavy chain NotI fragment are isolated asdescribed by Traver et al., Proc. Natl. Acad. Sci. USA, 86: 5898-5902(1989). The cloned NotI insert is isolated from high molecular weightyeast DNA by pulse field gel electrophoresis as described by M. Finney,op. cit. The DNA is condensed by the addition of 1 mM spermine andmicroinjected directly into the nucleus of single cell embryospreviously described. Alternatively, the DNA is isolated by pulsed fieldgel electrophoresis and introduced into ES cells by lipofection (Gnirkeet al., EMBO J. 10: 1629-1634 (1991)), or the YAC is introduced into EScells by spheroplast fusion.

Example 16 Discontinuous Genomic Heavy Chain Ig Transgene

An 85 kb SpeI fragment of human genomic DNA, containing V_(H) 6, Dsegments, J segments, the μ constant region and part of the γ constantregion, has been isolated by YAC cloning essentially as described inExample 1. A YAC carrying a fragment from the germline variable region,such as a 570 kb NotI fragment upstream of the 670-830 kb NotI fragmentdescribed above containing multiple copies of V₁ through V₅, is isolatedas described. (Berman et al. (1988), supra. detected two 570 kb NotIfragments, each containing multiple V segments.) The two fragments arecoinjected into the nucleus of a mouse single cell embryo as describedin Example 1.

Typically, coinjection of two different DNA fragments result in theintegration of both fragments at the same insertion site within thechromosome. Therefore, approximately 50% of the resulting transgenicanimals that contain at least one copy of each of the two fragments willhave the V segment fragment inserted upstream of the constant regioncontaining fragment. Of these animals, about 50% will carry out V to DJjoining by DNA inversion and about 50% by deletion, depending on theorientation of the 570 kb NotI fragment relative to the position of the85 kb SpeI fragment. DNA is isolated from resultant transgenic animalsand those animals found to be containing both transgenes by Southernblot hybridization (specifically, those animals containing both multiplehuman V segments and human constant region genes) are tested for theirability to express human immunoglobulin molecules in accordance withstandard techniques.

Example 17 Identification of Functionally Rearranged Variable RegionSequences in Transgenic B Cells

An antigen of interest is used to immunize (see Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y. (1988)) amouse with the following genetic traits: homozygosity at the endogenoushaving chain locus for a deletion of J_(H) (Examples 10); hemizygous fora single copy of unrearranged human heavy chain minilocus transgene(examples 5 and 14); and hemizygous for a single copy of a rearrangedhuman kappa light chain transgene (Examples 6 and 14).

Following the schedule of immunization, the spleen is removed, andspleen cells used to generate hybridomas. Cells from an individualhybridoma clone that secretes antibodies reactive with the antigen ofinterest are used to prepare genomic DNA. A sample of the genomic DNA isdigested with several different restriction enzymes that recognizeunique six base pair sequences, and fractionated on an agarose gel.Southern blot hybridization is used to identify two DNA fragments in the2-10 kb range, one of which contains the single copy of the rearrangedhuman heavy chain VDJ sequences and one of which contains the singlecopy of the rearranged human light chain VJ sequence. These twofragments are size fractionated on agarose gel and cloned directly intopUC18. The cloned inserts are then subcloned respectively into heavy andlight chain expression cassettes that contain constant region sequences.

The plasmid clone pγe1 (Example 12) is used as a heavy chain expressioncassette and rearranged VDJ sequences are cloned into the XhoI site. Theplasmid clone pCK1 is used as a light chain expression cassette andrearranged VJ sequences are cloned into the XhoI site. The resultingclones are used together to transfect SP₀ cells to produce antibodiesthat react with the antigen of interest (Co. et al., Proc. Natl. Acad.Sci. USA 88: 2869 (1991), which is incorporated herein by reference).

Alternatively, mRNA is isolated from the cloned hybridoma cellsdescribed above, and used to synthesize cDNA. The expressed human heavyand light chain VDJ and VJ sequence are then amplified by PCR and cloned(Larrick et al., Biol. Technology, 7: 934-938 (1989)). After thenucleotide sequence of these clones has been determined,oligonucleotides are synthesized that encode the same polypeptides, andsynthetic expression vectors generated as described by Queen et al.,Proc. Natl. Acad. Sci. USA., 84: 5454-5458 (1989).

Immunization of Transgenic Animals with Complex Antigens

The following experiment demonstrates that transgenic animals can besuccessfully immunized with complex antigens such as those on human redblood cells and respond with kinetics that are similar to the responsekinetics observed in normal mice.

Blood cells generally are suitable immunogens and comprise manydifferent types of antigens on the surface of red and white blood cells.

Immunization with Human Blood

Tubes of human blood from a single donor were collected and used toimmunize transgenic mice having functionally disrupted endogenous heavychain loci (J_(H) D) and harboring a human heavy chain minigeneconstruct (HC1); these mice are designated as line 112. Blood was washedand resuspended in 50 mls Hanks' and diluted to 1×10⁸ cells/ml 0.2 mls(2×10⁷ cells) were then injected interperitoneally using a 28 gaugeneedle and 1 cc syringe. This immunization protocol was repeatedapproximately weekly for 6 weeks. Serum titers were monitored by takingblood from retro-orbital bleeds and collecting serum and later testingfor specific antibody. A pre-immune bleed was also taken as a control.On the very last immunization, three days before these animals weresacrificed for serum and for hybridomas, a single immunization of 1′10⁷cells was given intravenously through the tail to enhance the productionof hybridomas.

TABLE 9 Animals Mouse ID Line Sex HC1-112 JHD 1 2343 112 M + ++ 2 2344112 M − + 3 2345 112 F − + 4 2346 112 F − ++ 5 2347 112 F − ++ 6 2348112 F + ++ 7 2349 112 F − + Mice # 2343 and 2348 have a desiredphenotype: human heavy chain mini-gene transgenic on heavy chainknock-out background.Generation of Hybridomas

Hybridomas were generated by fusing mouse spleen cells of approximately16 week-old transgenic mice (Table 9) that had been immunized asdescribed (supra) to a fusion partner consisting of the non-secretingHAT-sensitive myeloma cell line, X63 Ag8.653. Hybridoma clones werecultivated and hybridoma supernatants containing immunoglobulins havingspecific binding affinity for blood cell antigens were identified, forexample, by flow cytometry.

Flow Cytometry

Serum and hybridoma supernatants were tested using flow cytometry. Redblood cells from the donor were washed 4× in Hanks' balanced saltsolution and 50,000 cells were placed in 1.1 ml polypropylenemicrotubes. Cells were incubated with antisera or supernatant from thehybridomas for 30 minutes on ice in staining media (1×RPMI 1640 mediawithout phenol red or biotin (Irvine Scientific) 3% newborn calf serum,0.1% Na azide). Controls consisted of littermate mice with othergenotypes. Cells were then washed by centrifugation at 4° C. in SorvallRT600B for 5-10 minutes at 1000 rpm. Cells were washed two times andthen antibody detected on the cell surface with a fluorescent developingreagent. Two monoclonal reagents were used to test. One was aFITC-labeled mouse anti-human μ heavy chain antibody (Pharmagen, SanDiego, Calif.) and the other was a PE-labeled rat anti-mouse kappa lightchain (Becton-Dickenson, San Jose, Calif.). Both of these reagents gavesimilar results. Whole blood (red blood cells and white blood cells) andwhite blood cells alone were used as target cells. Both sets gavepositive results.

Serum of transgenic mice and littermate controls was incubated witheither red blood cells from the donor, or white blood cells from anotherindividual, washed and then developed with anti-human IgM FITC labeledantibody and analyzed in a flow cytometer. Results showed that serumfrom mice that are transgenic for the human mini-gene locus (mice 2343and 2348) show human IgM reactivity whereas all littermate animals(2344, 2345, 2346, 2347) do not. Normal mouse serum (NS) and phosphatebuffer saline (PBS) were used as negative controls. Red blood cells wereungated and white blood cells were gated to include only lymphocytes.Lines are drawn on the x and y axis to provide a reference. Flowcytometry was performed on 100 supernatants from fusion 2348. Foursupernatants showed positive reactivity for blood cell antigens.

Example 18 Reduction of Endogenous Mouse Immunoglobulin Expression byAntisense RNA

A. Vector for Expression of Antisense Ig Sequences

1. Construction of the Cloning Vector pGP1h

The vector pGP1b (referred to in a previous example) is digested withXhoI and BamHI and ligated with the following oligonucleotides:

(SEQ ID NO. 154) 5′-gat cct cga gac cag gta cca gat ctt gtg aat tcg-3′(SEQ ID NO. 155) 5′-tcg acg aat tca caa gat ctg gta cct ggt ctc gag-3′to generate the plasmid pGP1h. This plasmid contains a polylinker thatincludes the following restriction sites: NotI, EcoRI, BglII, Asp718,XhoI, BamHI, HindIII, NotI.Construction of pBCE1.

A 0.8 kb XbaI/BglII fragment of pVH251 (referred to in a previousexample), that includes the promoter leader sequence exon, first intron,and part of the second exon of the human VH-V family immunoglobulinvariable gene segment, was inserted into XbaI/BglII digested vectorpNNO3 to generate the plasmid pVH251.

The 2.2 kb BamHI/EcoRI DNA fragment that includes the coding exons ofthe human growth hormone gene (hGH; Seeburg, (1982) DNA 1: 239-249) iscloned into BglII/EcoRI digested pGH1h. The resulting plasmid isdigested with BamHI and the BamHI/BglII of pVH251N is inserted in thesame orientation as the hGH gene to generate the plasmid pvhgh.

A 0.9 kb XbaI fragment of mouse genomic DNA containing the mouse heavychain J-μ=0 intronic enhancer (Banerji et al., (1983) Cell 33: 729-740)was subcloned into pUC18 to generate the plasmid pJH22.1. This plasmidwas linearized with SphI and the ends filled in with klenow enzyme. Theklenow treated DNA was then digested with HindIII and a 1.4 kbMluI(klenow)/HindIII fragment of phage clone γ1.3 (previous example),containing the human heavy chain J-μintronic enhancer (Hayday et al.,(1984) Nature 307: 334-340), to it. The resulting plasmid, pMHE1,consists of the mouse and human heavy chain J-μ intron enhancers ligatedtogether into pUC18 such that they can be excised on a singleBamHI/HindIII fragment.

The BamHI/HindIII fragment of pMHE1 is cloned into BamHI/HindIII cutpvhgh to generate the B-cell expression vector pBCE1. This vector,depicted in FIG. 36, contains unique XhoI and Asp718 cloning sites intowhich antisense DNA fragments can be cloned. The expression of theseantisense sequences is driven by the upstream heavy chainpromoter-enhancer combination the downstream hGH gene sequences providepolyadenylation sequences in addition to intron sequences that promotethe expression of transgene constructs. Antisense transgene constructsgenerated from pBCE1 can be separated from vector sequences by digestionwith NotI.

B. An IgM Antisense Transgene Construct.

The following two oligonucleotides:

(SEQ ID NO. 156) 5′-cgc ggt acc gag agt cag tcc ttc cca aat gtc-3′ (SEQID NO. 157) 5′-cgc ctc gag aca gct gga atg ggc aca tgc aga-3′are used as primers for the amplification of mouse IgM constant regionsequences by polymerase chain reaction (PCR) using mouse spleen cDNA asa substrate. The resulting 0.3 kb PCR product is digested with Asp718and XhoI and cloned into Asp718/XhoI digested pBCE1 to generate theantisense transgene construct pMAS1. The purified NotI insert of pMAS1is microinjected into the pronuclei of half day mouse embryos—alone orin combination with one or more other transgene constructs—to generatetransgenic mice. This construct expresses an RNA transcript in B-cellsthat hybridizes with mouse IgM mRNA, thus down-regulating the expressionof mouse IgM protein. Double transgenic mice containing pMAS1 and ahuman heavy chain transgene minilocus such as pHC1 (generated either bycoinjection of both constructs or by breeding of singly transgenic mice)will express the human transgene encoded Ig receptor on a higherpercentage of B-cell than mice transgenic for the human heavy chainminilocus alone. The ratio of human to mouse Ig receptor expressingcells is due in part to competition between the two populations forfactors and cells that promoter B-cell differentiation and expansion.Because the Ig receptor plays a key role in B-cell development, mouse Igreceptor expressing B-cells that express reduced levels of IgM on theirsurface (due to mouse Ig specific antisense down-regulation) duringB-cell development will not compete as well as cells that express thehuman receptor.C. An IgKappa Antisense Transgene Construct.

The following two oligonucleotides:

(SEQ ID NO. 158) 5′-cgc ggt acc gct gat gct gca cca act gta tcc-3′ (SEQID NO. 159) 5′-cgc ctc gag cta aca ctc att cct gtt gaa gct-3′are used as primers for the amplification of mouse IgKappa constantregion sequences by polymerase chain reaction (PCR) using mouse spleencDNA as a substrate. The resulting 0.3 kb PCR product is digested withAsp718 and XhoI and cloned into Asp718/XhoI digested pBCE1 to generatethe antisense transgene construct pKAS1. The purified NotI insert ofpKAS1 is microinjected into the pronuclei of half day mouseembryos—alone or in combination with one or more other transgeneconstructs—to generate transgenic mice. This construct expresses an RNAtranscript in B-cells that hybridizes with mouse IgK mRNA, thusdown-regulating the expression of mouse IgK protein as described abovefor pMAS1.

Example 19

This example demonstrates the successful immunization and immuneresponse in a transgenic mouse of the present invention.

Immunization of Mice

Keyhole limpet hemocyanin conjugated with greater than 400 dinitrophenylgroups per molecule (Calbiochem, La Jolla, Calif.) (KLH-DNP) was alumprecipitated according to a previously published method (PracticalImmunology, L. Hudson and F. C. Hay, Blackwell Scientific (Pubs.), p. 9,1980). Four hundred μg of alum precipitated KLH-DNP along with 100 μgdimethyldioctadecyl Ammonium Bromide in 100 μL of phosphate bufferedsaline (PBS) was injected intraperitoneally into each mouse. Serumsamples were collected six days later by retro-orbital sinus bleeding.

Analysis of Human Antibody Reactivity in Serum

Antibody reactivity and specificity were assessed using an indirectenzyme-linked immunosorbent assay (ELISA). Several target antigens weretested to analyze antibody induction by the immunogen. Keyhole limpethemocyanin (Calbiochem) was used to identify reactivity against theprotein component, bovine serum albumin-DNP for reactivity against thehapten and/or modified amino groups, and KLH-DNP for reactivity againstthe total immunogen. Human antibody binding to antigen was detected byenzyme conjugates specific for IgM and IgG sub-classes with no crossreactivity to mouse immunoglobulin. Briefly, PVC microtiter plates werecoated with antigen drying overnight at 37° C. of 5 μg/mL protein inPBS. Serum samples diluted in PBS, 5% chicken serum, 0.5% Tween-20 wereincubated in the wells for 1 hour at room temperature, followed byanti-human IgG Fc and IgG F(ab′)-horseradish peroxidase or anti-humanIgM Fc-horseradish peroxidase in the same diluent. After 1 hour at roomtemperature enzyme activity was assessed by addition of ABTS substrate(Sigma, St. Louis, Mo.) and read after 30 minutes at 415-490 nm.

Human Heavy Chain Participation in Immune Response in Transgenic Mice

FIGS. 37A-37D illustrate the response of three mouse littermates toimmunization with KLH-DNP. Mouse number 1296 carried the human IgM andIgG unrearranged transgene and was homozygous for mouse Ig heavy chainknockout. Mouse number 1299 carried the transgene on a non-knockoutbackground, while mouse 1301 inherited neither of these sets of genes.Mouse 1297, another littermate, carried the human transgene and washemizygous with respect to mouse heavy chain knockout. It was includedas a non-immunized control.

The results demonstrate that both human IgG and IgM responses weredeveloped to the hapten in the context of conjugation to protein. HumanIgM also developed to the KLH molecule, but no significant levels ofhuman IgG were present at this time point. In pre-immunization serumsamples from the same mice, titers of human antibodies to the sametarget antigens were insignificant.

Example 20

This example demonstrates the successful immunization with a humanantigen and immune response in a transgenic mouse of the presentinvention, and provides data demonstrating that nonrandom somaticmutation occurs in the variable region sequences of the human transgene.

Demonstration of Antibody Responses Comprising Human ImmunoglobulinHeavy Chains Against a Human Glycoprotein Antigen

Transgenic mice used for the experiment were homozygous for functionallydisrupted murine immunoglobulin heavy chain loci produced byintroduction of a transgene at the joining (J) region (supra) resultingin the absence of functional endogenous (murine) heavy chain production.The transgenic mice also harbored at least one complete unrearrangedhuman heavy chain mini-locus transgene, (HC1, supra), which included asingle functional V_(H) gene (V_(H) 251), human μ constant region gene,and human γ1 constant region gene. Transgenic mice shown to expresshuman immunoglobulin transgene products (supra) were selected forimmunization with a human antigen to demonstrate the capacity of thetransgenic mice to make an immune response against a human antigenimmunization. Three mice of the HC1-26 line and three mice of the HC1-57line (supra) were injected with human antigen.

One hundred μg of purified human carcinoembryonic antigen (CEA)insolubilized on alum was injected in complete Freund's adjuvant on Day0, followed by further weekly injections of alum-precipitated CEA inincomplete Freund's adjuvant on Days 7, 14, 21, and 28. Serum sampleswere collected by retro-orbital bleeding on each day prior to injectionof CEA. Equal volumes of serum were pooled from each of the three micein each group for analysis.

Titres of human μ chain-containing immunoglobulin and human γchain-containing immunoglobulin which bound to human CEA immobilized onmicrotitre wells were determined by ELISA assay. Results of the ELISAassays for human μ chain-containing immunoglobulins and human γchain-containing immunoglobulins are shown in FIGS. 38 and 39,respectively. Significant human μ chain Ig titres were detected for bothlines by Day 7 and were observed to rise until about Day 21. For human γchain Ig, significant titres were delayed, being evident first for lineHC1-57 at Day 14, and later for line HC1-26 at Day 21. Titres for humany chain Ig continued to show an increase over time during the course ofthe experiment. The observed human μ chain Ig response, followed by aplateau, combined with a later developing γ chain response whichcontinues to rise is characteristic of the pattern seen with affinitymaturation. Analysis of Day 21 samples showed lack of reactivity to anunrelated antigen, keyhole limpet hemocyanin (KLC), indicating that theantibody response was directed against CEA in a specific manner.

These data indicate that animals transgenic for human unrearrangedimmunoglobulin gene loci: (1) can respond to a human antigen (e.g., thehuman glycoprotein, CEA), (2) can undergo isotype switching (“classswitching) as exemplified by the observed μ to γ class switch, and (3)exhibit characteristics of affinity maturation in their humoral immuneresponses. In general, these data indicate: (1) the human Ig transgenicmice have the ability to induce heterologous antibody production inresponse to a defined antigen, (2) the capacity of a single transgeneheavy chain variable region to respond to a defined antigen, (3)response kinetics over a time period typical of primary and secondaryresponse development, (4) class switching of a transgene-encoded humoralimmune response from IgM to IgG, and (5) the capacity of transgenicanimal to produce human-sequence antibodies against a human antigen.

Demonstration of Somatic Mutation in a Human Heavy Chain TransgeneMinilocus.

Line HC1-57 transgenic mice, containing multiple copies of the HC1transgene, were bred with immunoglobulin heavy chain deletion mice toobtain mice that contain the HC1 transgene and contain disruptions atboth alleles of the endogenous mouse heavy chain (supra). These miceexpress human mu and gammal heavy chains together with mouse kappa andlambda light chains (supra). One of these mice was hyperimmunizedagainst human carcinoembryonic antigen by repeated intraperitonealinjections over the course of 1.5 months. This mouse was sacrificed andlymphoid cells isolated from the spleen, inguinal and mesenteric lymphnodes, and peyers patches. The cells were combined and total RNAisolated. First strand cDNA was synthesized from the RNA and used as atemplate for PCR amplification with the following 2 oligonucleotideprimers:

149 (SEQ ID NO. 82) 5′-cta gct cga gtc caa gga gtc tgt gcc gag gtg cagctg(g/a/t/c)-3′ 151 (SEQ ID NO. 84) 5′-ggc gct cga gtt cca cga cac cgtcac cgg ttc-3′

These primers specifically amplify VH251/gammal cDNA sequences. Theamplified sequences were digested with XhoI and cloned into the vectorpNN03. DNA sequence from the inserts of 23 random clones is shown inFIG. 40; sequence variations from germline sequence are indicated, dotsindicate sequence is identical to germline. Comparison of the cDNAsequences with the germline sequence of the VH251 transgene reveals that3 of the clones are completely unmutated, while the other 20 clonescontain somatic mutations. One of the 3 non-mutated sequences is derivedfrom an out-of-frame VDJ joint. Observed somatic mutations at specificpositions of occur at similar frequencies and in similar distributionpatterns to those observed in human lymphocytes (Cai et al. (1992) J.Exp. Med. 176: 1073, incorporated herein by reference). The overallfrequency of somatic mutations is approximately 1%; however, thefrequency goes up to about 5% within CDR1, indicating selection foramino acid changes that affect antigen binding. This demonstratesantigen driven affinity maturation of the human heavy chain sequences.

Example 21

This example demonstrates the successful formation of a transgene byco-introduction of two separate polynucleotides which recombine to forma complete human light chain minilocus transgene.

Generation of an Unrearranged Light Chain Minilocus Transgene byCo-Injection of Two Overlapping DNA Fragments

1. Isolation of Unrearranged Functional V_(κ) Gene Segments vk65.3,vk65.5, vk65.8 and vk65.15

The V_(κ) specific oligonucleotide, oligo-65 (5′-agg ttc agt ggc agt gggtct ggg aca gac ttc act ctc acc atc agc-3′) (SEQ ID NO.153), was used toprobe a human placental genomic DNA library cloned into the phage vectorλEMBL3/SP6/T7 (Clonetech Laboratories, Inc., Palo Alto, Calif.). DNAfragments containing V_(κ) segments from positive phage clones weresubcloned into plasmid vectors. Variable gene segments from theresulting clones are sequenced, and clones that appear functional wereselected. Criteria for judging functionality include: open readingframes, intact splice acceptor and donor sequences, and intactrecombination sequence. DNA sequences of 4 functional V_(κ) genesegments (vk65.3, vk65.5, vk65.8, and vk65.15) from 4 different plasmidclones isolated by this procedure are shown in FIGS. 41-44. The fourplasmid clones, p65.3f, p65.5 g1, p65.8, and p65.15f, are describedbelow.

(1 a) p65.3f

A 3 kb Xba fragment of phage clone λ65.3 was subcloned into pUC19 sothat the vector derived SalI site was proximal to the 3′ end of theinsert and the vector derived BamHI site 5′. The 3 kb BamHI/SalI insertof this clone was subcloned into pGP1f to generate p65.3f.

(1 b) p65.5g1

A 6.8 kb EcoRI fragment of phage clone λ65.5 was subcloned into pGP1f sothat the vector derived XhoI site is proximal to the 5′ end of theinsert and the vector derived SalI site 3′. The resulting plasmid isdesignated p65.5g1.

(1 c) p65.8

A 6.5 kb HindIII fragment of phage clone λ65.8 was cloned into pSP72 togenerate p65.8.

(1 d) p65.15f

A 10 kb EcoRI fragment of phage clone λ65.16 was subcloned into pUC18 togenerate the plasmid p65.15.3. The V_(κ) gene segment within the plasmidinsert was mapped to a 4.6 kb EcoRI/HindIII subfragment, which wascloned into pGP1f. The resulting clone, p65.15f, has unique XhoI andSalI sites located at the respective 5′ and 3′ ends of the insert.

2. pKV4

The XhoI/SalI insert of p65.8 was cloned into the XhoI site of p65.15fto generate the plasmid pKV2. The XhoI/SalI insert of p65.5g1 was clonedinto the XhoI site of pKV2 to generate pKV3. The XhoI/SalI insert ofpKV3 was cloned into the XhoI site of p65.3f to generate the plasmidpKV4. This plasmid contains a single 21 kb XhoI/SalI insert thatincludes 4 functional V_(κ)0 gene segments. The entire insert can alsobe excised with NotI.

3. pKC1B

(3a) pKcor

Two XhoI fragments derived from human genomic DNA phage λ clones weresubcloned into plasmid vectors. The first, a 13 kb J_(κ)2-J_(κ)5/C_(κ)containing fragment, was treated with Klenow enzyme and cloned intoHindIII digested, Klenow treated, plasmid pGP1d. A plasmid clone (pK-31)was selected such that the 5′ end of the insert is adjacent to thevector derived ClaI site. The second XhoI fragment, a 7.4 kb piece ofDNA containing J_(κ)1 was cloned into XhoI/SalI-digested pSP72, suchthat the 3′ insert XhoI site was destroyed by ligation to the vectorSalI site. The resulting clone, p36.2s, includes an insert derived ClaIsite 4.5 kb upstream of J_(κ)1 and a polylinker derived ClaI sitedownstream in place of the naturally occurring XhoI site between J_(κ)1and J_(κ)2. This clone was digested with ClaI to release a 4.7 kbfragment which was cloned into ClaI digested pK-31 in the correct 5′ to3′ orientation to generate a plasmid containing all 5 human J_(κ)segments, the human intronic enhancer human C_(κ), 4.5 kb of 5′ flankingsequence, and 9 kb of 3′ flanking sequence. This plasmid, pKcor,includes unique flanking XhoI and SalI sites on the respective 5′ and 3′sides of the insert.

(3 b) pKcorB

A 4 kb BamHI fragment containing the human 3′ kappa enhancer (Judde,J.-G. and Max, E. E. (1992) Mol. Cell. Biol. 12: 5206, incorporatedherein by reference) was cloned into pGP1f such that the 5′ end isproximal to the vector XhoI site. The resulting plasmid, p24Bf, was cutwith XhoI and the 17.7 kb XhoI/SalI fragment of pKcor cloned into it inthe same orientation as the enhancer fragment. The resulting plasmid,pKcorB, includes unique XhoI and SalI sites at the 5′ and 3′ ends of theinsert respectively.

(3 c) pKC1B

The XhoI/SalI insert of pKcorB was cloned into the SalI site of p65.3fto generate the light-chain minilocus-transgene plasmid pKC1B. Thisplasmid includes a single functional human V_(κ) segment, all 5 humanJ_(κ) segments, the human intronic enhancer, human C_(κ), and the human3′ kappa enhancer. The entire 25 kb insert can be isolated by NotIdigestion.

4. Co4

The two NotI inserts from plasmids pKV4 and pKC1B were mixed at aconcentration of 2.5 μg/ml each in microinjection buffer, andco-injected into the pronuclei of half day mouse embryos as described inprevious examples. Resulting transgenic animals contain transgeneinserts (designated Co4, product of the recombination shown in FIG. 45)in which the two fragments co-integrated. The 3′ 3 kb of the pKV4 insertand the 5′ 3 kb of the pKC1B insert are identical. Some of theintegration events will represent homologous recombinations between thetwo fragments over the 3 kb of shared sequence. The Co4 locus willdirect the expression of a repertoire of human sequence light chains ina transgenic mouse.

Example 22

This example demonstrates the successful production of a murinehybridoma clone secreting a monoclonal antibody reactive with a specificimmunogen, wherein the monoclonal antibody comprises a humanimmunoglobulin chain encoded by a human Ig transgene.

Generation of Monoclonal Antibodies Incorporating Human Heavy ChainTransgene Product

1. Immunization of Mouse Harboring Human Heavy Chain Transgene

A mouse containing a human heavy chain encoding transgene and homozygousfor knockout (i.e., functional disruption) of the endogenous heavy chainlocus (see, EXAMPLE 20, supra) was immunized with purified human CEA,and spleen cells were subsequently harvested after a suitable immuneresponse period. The murine spleen cells were fused with mouse myelomacells to generate hybridomas using conventional techniques (see, Kohlerand Milstein, Eur. J. Immunol., 6:511-519 (1976); Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y. (1988)). Themouse used for immunization contained a human unrearranged heavy chainminilocus transgene which comprised a single functional V_(H) gene(V_(H) 251), human D and J segments, human p constant region, and humanγ1 constant region genes. The transgenic line from which it originatedwas designated HC1-57 (supra).

One hundred μg of purified human carcinoembryonic antigen (CEA) (CyrstalChem, Chicago, Ill. or Scripps Labs, San Diego, Calif.) insolubilized onalum was injected in complete Freund's adjuvant on Day 0, followed byfurther weekly injections of alum-precipitated CEA in incompleteFreund's adjuvant on Days 7, 14, 21, and 28. An additional 20 μg ofsoluble CEA was administered intravenously on Day 83, followed by 50 μgalum-precipitated CEA in incomplete Freund's adjuvant on Day 92. Humanheavy chain responses to CEA were confirmed in serum samples prior tofusion of spleen cells with myeloma cells. The animal was sacrificed onDay 95, the spleen removed and fused with P3×63-Ag8.653 mouse myelomacells (ATCC CRL 1580, American Type Culture Collection, Rockville, Md.)using polyethylene glycol. Two weeks later, supernates from fusion wellswere screened for the presence of antibodies specifically reactive withCEA, and which contained human heavy chain μ or γ constant regionepitopes by ELISA. Briefly, purified human CEA was coated onto PVCmicrotitre plates at 2.5 μg/ml, and incubate with culture supernatediluted 1:4 or 1:5 in PBS, 0.5% Tween-20, 5% chicken serum. Plates werewashed, followed by addition of horseradish peroxidase-conjugated goatantiserum specific for human IgG Fc or rabbit antiserum specific forhuman IgM Fc5Mu (Jackson ImmunoResearch, West Grove, Pa.). Presence ofconjugate bound to captured antibody was determined, after furtherwashing, by the addition of ABTS substrate. Two independent fusion wellswere found to contain antibody with substantial binding to CEA. Aftercloning, both hybridomas were found to be positive for the presence ofhuman μ chain and murine κ chain by ELISA. No mouse IgG or IgM weredetected using similar assays.

Subcloning of the two independent parent hybridomas resulted in twoclones, designated 92-09A-4F7-A5-2 and 92-09A-1D7-1-7-1. Both lines weredeposited with the ATCC Patent Culture Depository under the BudapestTreaty and were assigned ATCC Designation HB 11307 and HB 11308,respectively. Culture supernatants from these cell lines were assessedfor specificity by testing for reactivity to several purified targetproteins using ELISA. As shown in FIG. 46, ELISA assays for determiningthe reactivity of the monoclonal antibodies to various antigensdemonstrate that only CEA and the CEA-related antigen NCA-2 showsignificant reactivity, indicating the development of a restrictedreactivity for the variable regions of the heterohybrid immunoglobulinmolecules.

Example 23

This example demonstrates that a rearranged human VDJ gene encoded by ahuman Ig minilocus transgene may be transcribed as a transcript whichincludes an endogenous Ig constant region gene, for example by themechanism of trans-switching, to encode a chimeric human/mouse Ig chain.

Identification of Trans-Switch Transcripts Encoding Chimeric Human-MouseHeavy Chains

RNA was isolated from a hyperimmunized HC1 line 57 transgenic mousehomozygous for the endogenous heavy chain J segment deletion (supra).cDNA was synthesized according to Taylor et al. (1993) Nucleic AcidsRes. 20: 6287, incorporated herein by reference, and amplified by PCRusing the following two primers:

o-149 (human VH251): (SEQ ID NO. 82) 5′-CTA GCT CGA GTC CAA GGA GTC TGTGCC GAG GTG CAG CTG(G, A, T, C)-3′ o-249 (mouse gamma): (SEQ ID NO. 160)5′-GGC GCT CGA GCT GGA CAG GG(A/C) TCC A(G/T)A GTT CCA-3′

Oligonucleotide o-149 is specific for the HC1-encoded variable genesegment V_(H) 251, while o-249 hybridizes to both mouse and human gammasequences with the following order of specificities: mouse γ1=mouseγ2b=mouse γ3>mouse γ2a>>human γ1. DNA sequences from 10 randomly chosenclones generated from the PCR products was determined and is shown inFIG. 47. Two clones comprised human VDJ and mouse γ1; four clonescomprised human VDJ and mouse γ2b; and four clones comprised human VDJand mouse γ3. These results indicate that in a fraction of thetransgenic B cells, the transgene-encoded human VDJ recombined into theendogenous murine heavy chain locus by class switching or an analogousrecombination.

Example 24

This example describes a method for screening a pool of hybridomas todiscriminate clones which encode chimeric human/mouse Ig chains fromclones which encode and express a human Ig chain. For example, in a poolof hybridoma clones made from a transgenic mouse comprising a human Igheavy chain transgene and homozygous for a J region-disrupted endogenousheavy chain locus, hybridoma clones encoding trans-switched humanVDJ-murine constant region heavy chains may be identified and separatedfrom hybridoma clones expressing human VDJ-human constant region heavychains.

Screening Hybridomas to Eliminate Chimeric Ig Chains

The screening process involves two stages, which may be conducted singlyor optionally in combination: (1) a preliminary ELISA-based screen, and(2) a secondary molecular characterization of candidate hybridomas.Preferably, a preliminary ELISA-based screen is used for initialidentification of candidate hybridomas which express a human VDJ regionand a human constant region.

Hybridomas that show positive reactivity with the antigen (e.g., theimmunogen used to elicit the antibody response in the transgenic mouse)are tested using a panel of monoclonal antibodies that specificallyreact with mouse μ, γ, κ, and λ, and human μ, γ, and κ. Only hybridomasthat are positive for human heavy and light chains, as well as negativefor mouse chains, are identified as candidate hybridomas that expresshuman immunoglobulin chains. Thus, candidate hybridomas are shown tohave reactivity with specific antigen and to possess epitopescharacteristic of a human constant region.

RNA is isolated from candidate hybridomas and used to synthesize firststrand cDNA. The first strand cDNA is then ligated to a uniquesingle-stranded oligonucleotide of predetermined sequence (oligo-X)using RNA ligase (which ligates single-stranded DNA). The ligated cDNAis then amplified in two reactions by PCR using two sets ofoligonucleotide primers. Set H (heavy chain) includes an oligo thatspecifically anneals to either human μ or human γ1 (depending on theresults of the ELISA) and an oligo that anneals to the oligo-X sequence.This prevents bias against detection of particular V segments, includingmouse V segments that may have trans-rearranged into the humanminilocus. A second set of primers, Set L (light chain), includes anoligo that specifically anneals to human κ and an oligo that annealsspecifically to oligo-X. The PCR products are molecularly cloned and theDNA sequence of several are determined to ascertain whether thehybridoma is producing a unique human antibody on the basis of sequencecomparison to human and murine Ig sequences.

Example 25

This example demonstrates production of a transgenic mouse harboring ahuman light chain (κ) minilocus.

Human κ Minilocus Transgenic Mice

KC1

A 13 kb XhoI Jκ2-Kκ containing fragment from a phage clone (isolatedfrom a human genomic DNA phage library by hybridization to a κ specificoligonucleotide, e.g., supra) was treated with Klenow enzyme and clonedinto the Klenow treated HindIII site of pGP1d to produce pK-31. Thisdestroyed the insert XhoI sites and positioned the unique polylinkerderived XhoI site at the 5′ end next to Jκ2. A unique polylinker derivedClaI site is located between this XhoI site and the inset sequences,while a unique polylinker derived SalI site is located at the 3′ end ofthe insert. A 7.5 kb XhoI fragment, containing Jκ1 and upstreamsequences, was also isolated from a human genomic DNA phage clone(isolated from a human genomic DNA phage library by hybridization to a κspecific oligonucleotide, e.g. supra). This 7.5 kb XhoI fragment wascloned into the SalI site of pSP72 (Promega, Madison, Wis.), thusdestroying both XhoI sites and positioning a polylinker ClaI site 3′ ofJκ1. Digestion of the resulting clone with ClaI released a 4.7 kbfragment containing Jk1 and 4.5 kb of upstream sequences. This 4.7 kbfragment was cloned into the ClaI site of pK-31 to create pKcor. Theremaining unique 5′ XhoI site is derived from polylinker sequences. A6.5 kb XhoI/SalI DNA fragment containing the unrearranged human VκIIIgene segment 65.8 (plasmid p65.8, EXAMPLE 21) was cloned into the XhoIsite of pKcor to generate the plasmid pKC1. The NotI insert of pKC1 wasmicroinjected into ½ day mouse embryos to generate transgenic mice. Twoindependent pKC1 derived transgenic lines were established and used tobreed mice containing both heavy and light chain miniloci. These lines,KC1-673 and KC1-674, were estimated by Southern blot hybridization tocontain integrations of approximately 1 and 10-20 copies of thetransgenes respectively.

KC1e

The plasmid pMHE1 (EXAMPLES 13 and 18) was digested with BamHI andHindIII to excise the 2.3 kb insert containing both the mouse and humanheavy chain J-μ intronic enhancers. This fragment was Klenow treated,ligated to SalI linkers (New England Biolabs, Beverly, Mass.), andcloned into the unique 3′ SalI site of pKC1 to generate the plasmidpKC1e. The NotI insert of pKC1e was microinjected into ½ day mouseembryos to generate transgenic mice. Four independent pKC1e derivedtransgenic lines were established and used to breed mice containing bothheavy and light chain miniloci. These lines, KC1e-1399, KC1e-1403,KC1e-1527, and KC1e-1536, were estimated by Southern blot hybridizationto contain integrations of approximately 20-50, 5-10, 1-5, and 3-5copies of the transgene, respectively.

PKC2

A 6.8 kb XhoI/SalI DNA fragment containing the unrearranged human VκIIIgene segment 65.5 (plasmid p65.5g1, EXAMPLE 21) was cloned into theunique 5′ XhoI site of pKC1 to generate the plasmid pKC2. This minilocustransgene contains two different functional VκIII gene segments. TheNotI insert of pKC2 was microinjected into ½ day mouse embryos togenerate transgenic mice. Five independent pKC2 derived transgenic lineswere established and used to breed mice containing both heavy and lightchain miniloci. These lines, KC2-1573, KC2-1579, KC2-1588, KC2-1608, andKC2-1610, were estimated by Southern blot hybridization to containintegrations of approximately 1-5, 10-50, 1-5, 50-100, and 5-20 copiesof the transgene, respectively.

Example 26

This example shows that transgenic mice bearing the human κ transgenecan make an antigen-induced antibody response forming antibodiescomprising a functional human κ chain.

Antibody Responses Associated with Human Ig κ Light Chain

A transgenic mouse containing the HC1-57 human heavy chain and KC1ehuman κ transgenes was immunized with purified human soluble CD4 (ahuman glycoprotein antigen). Twenty μg of purified human CD4 (NENResearch products, Westwood, Mass.) insolublized by conjugation topolystyrene latex particles (Polysciences, Warrington, Pa.) was injectedintraperitoneally in saline with dimethyldioctadecyl ammonium bromide(Calbiochem, San Diego, Calif.) on Day 0, followed by further injectionson Day 20 and Day 34.

Retro-orbital bleeds were taken on Days 25 and 40, and screened for thepresence of antibodies to CD4, containing human IgM or human IgG heavychain by ELISA. Briefly, purified human CD4 was coated onto PVCmicrotitre plates at 2.5 μg/ml and incubated with culture supernatediluted 1:4/1:5 in PBS, 0.5% Tween-20, 5% chicken serum. Plates werewashed, followed by addition of horseradish peroxidase-conjugated goatantiserum specific for human IgG Fc or rabbit antiserum specific forhuman IgM Fc5Mu (Jackson ImmunoResearch, West Grove, Pa.). Presence ofconjugate bound to captured antibody was determined after furtherwashing by addition of ABTS substrate. Human μ reactive with antigen wasdetected in both bleeds, while there was essentially undetectable γreactivity. The Day 40 sample was also tested for antigen-reactive humanκ chain using the same assay with goat anti-human κ peroxidase conjugate(Sigma, St. Louis, Mo.). CD4-binding κ reactivity was detected at thistime point. The assay results are shown in FIG. 48.

Example 27

This example shows the successful generation of mice which arehomozygous for functionally disrupted murine heavy and light chain loci(heavy chain and κ chain loci) and which concomitantly harbor a humanheavy chain transgene and a human light chain transgene capable ofproductively rearranging to encode functional human heavy chains andfunctional human light chains. Such mice are termed “0011” mice,indicating by the two 0's in the first two digits that the mice lackfunctional heavy and light chain loci and indicating by the 1's in thesecond two digits that the mice are hemizygous for a human heavy chaintransgene and a human light chain transgene. This example shows thatsuch 0011 mice are capable of making a specific antibody response to apredetermined antigen, and that such an antibody response can involveisotype switching.

0011/0012 Mice: Endogenous Ig Knockout+Human Ig Transgenes

Mice which were homozygous for a functionally disrupted endogenous heavychain locus lacking a functional J_(H) region (designated JHD++ orJHA++) and also harboring the human HC1 transgene, such as the HC1-26transgenic mouse line described supra, were interbred with micehomozygous for a functionally disrupted endogenous kappa chain locuslacking a functional J_(H) region (designated here as JKD++ or JKδ++;see Example 9) to produce mice homozygous for functionally disruptedheavy chain and kappa chain loci (heavy chain/kappa chain knockouts),designated as JHD++/JKD++ and containing a HC1 transgene. Such mice wereproduced by interbreeding and selected on the basis of genotype asevaluated by Southern blot of genomic DNA. These mice, designatedHC1-26+/JKD++/JHD++mice, were interbred with mice harboring a humankappa chain transgene (lines KC2-1610, KC1e-1399, and KC1e-1527; seeExample 25), and Southern blot analysis of genomic DNA was used toidentify offspring mice homozygous for i functionally disrupted heavyand light chain loci and also hemizygous for the HC1 transgene and theKC2 or KC1e transgene. Such mice are designated by numbers and wereidentified as to their genotype, with the following abbreviations:HC1-26+ indicates hemizygosity for the HC1-26 line human heavy chainminilocus transgene integration; JHD++ indicates homozygosity for J_(H)knockout; JKD++ indicates homozygosity for J_(K) knockout; KC2-1610+indicates hemizygosity for a KC2 human κ transgene integrated as in lineKC2-1610; KC1e-1527+ indicates hemizygosity for a KC1e human κ transgeneintegrated as in line KC1e-1527; KC1e-1399+ indicates hemizygosity for aKC1e human κ transgene integrated as in line KC1e-1399.

The resultant individual offspring were each given a numericaldesignation (e.g., 6295, 6907, etc.) and each was evaluated for thepresence of J_(H) knockout alleles, J_(K) knockout alleles, HC1-26transgene, and κ transgene (KC2 or KC1e) and determined to be eitherhemizygous (+) or homozygous (++) at each locus. Table 10 shows thenumber designation, sex, and genotypes of several of the offspring mice.

TABLE 10 ID No. Sex Ig Code Genotype 6295 M 0011 HC1-26+; JHD++; JKD++;KC2-1610+ 6907 M 0011 HC1-26+; JHD++; JKD++; KC1e-1527+ 7086 F 0011HC1-26+; JHD++; JKD++; KC1e-1399+ 7088 F 0011 HC1-26+; JHD++; JKD++;KC1e-1399+ 7397 F 0011 HC1-26+; JHD++; JKD++; KC1e-1527+ 7494 F 0012HC1-26+; JHD++; JKD++; KC2-1610++ 7497 M 0011 HC1-26+; JHD++; JKD++;KC1e-1399+ 7648 F 0011 HC1-26+; JHD++; JKD++; KC2-1610+ 7649 F 0012HC1-26+; JHD++; JKD++; KC2-1610++ 7654 F 0011 HC1-26+; JHD++; JKD++;KC2-1610+ 7655 F 0011 HC1-26+; JHD++; JKD++; KC2-1610+ 7839 F 0011HC1-26+; JHD++; JKD++; KC1e-1399+ 7656 F 0001 HC1-26−; JHD++; JKD++;KC2-1610+ 7777 F 1100 Co1-2141−; JHD+; JKD+

We removed spleens from three 6 week old female mice. Mouse #7655 wasdetermined by Southern blot hybridization to be hemizygous for the HC1(line 26) and KC2 (line 1610) transgene integrations, and homozygous forthe JHΔ and JκΔ targeted deletions of the mouse μ and κJ regions. Mouse#7656 was determined by Southern blot hybridization to be hemizygous forthe KC2 (line 1610) transgene integration and homozygous for the JHΔ andJκΔ targeted deletions of the mouse μ and κJ regions. Mouse 1 7777 wasdetermined by Southern blot hybridization to be hemizygous for the JHΔand JκΔ targeted deletions of the mouse μ and κJ regions. Because of therecessive nature of these deletions, this mouse should be phenotypicallywild-type.

Expression of Endogenous Ig Chains in 0011 Mice

FACS analysis using a panel of antibodies reactive with either human μ,mouse μ, human κ, mouse κ, or mouse λ was used to sort lymphocytesexplanted from (1) a wildtype mouse (7777), (2) a 0001 mouse homozygousfor heavy chain and kappa knockout alleles and harboring a human lightchain transgene (7656), and (3) a 0011 mouse homozygous for heavy chainand kappa knockout alleles and harboring a human light chain transgeneand a human heavy chain transgene (7655).

We prepared single cell suspensions from spleen and lysed the red cellswith NH₄Cl, as described by Mishell and Shiigi (Mishell, B. B. & Shiigi,S. M. (eds) Selected Methods in Cellular Immunology. W.H. Freeman & Co.,New York, 1980). The lymphocytes are stained with the followingreagents: propidium iodide (Molecular Probes, Eugene, Oreg.), FITCconjugated anti-human IgM (clone G20-127; Pharmingen, San Diego,Calif.), FITC conjugated anti-mouse IgM (clone R6-60.2; Pharmingen, SanDiego, Calif.), phycoerythrin conjugated anti-human Igκ (clone HP6062;CalTag, South San Francisco, Calif.), FITC conjugated anti-mouse Igλ(clone R26-46; Pharmingen, San Diego, Calif.) FITC conjugated anti-mouseB220 (clone RA3-6B2; Pharmingen, San Diego, Calif.), and Cy-Chromeconjugated anti-mouse B220 (clone RA3-6B2; Pharmingen, San Diego,Calif.). We analyzed the stained cells using a FACScan flow cytometerand LYSIS II software (Becton Dickinson, San Jose, Calif.). Macrophagesand residual red cells are excluded by gating on forward and sidescatter. Dead cells are excluded by gating out propidium iodide positivecells. The flow cytometric data in FIGS. 49 and 50 confirms the Southernblot hybridization data and demonstrates that mouse #7655 expresses bothhuman μ and human κ and relatively little if any mouse μ or mouse κ.Nevertheless a significant fraction of the B cells (about 70-80%) appearto express hybrid Ig receptors consisting of human heavy and mouse λlight chains.

FIG. 49 shows the relative distribution of B cells expressing human μ ormouse μ on the cell surface; 0011 mouse (7655) lymphocytes are positivefor human μ but relatively lack mouse μ; 0001 mouse (7656) lymphocytesdo not express much human μ or mouse μ; wildtype mouse (7777)lymphocytes express mouse μ but lack human μ.

FIG. 50 shows the relative distribution of B cells expressing human κ ormouse κ on the cell surface; 0011 mouse (7655) lymphocytes are positivefor human κ but relatively lack mouse κ; 0001 mouse (7656) lymphocytesdo not express much human κ or mouse κ; wildtype mouse (7777)lymphocytes express mouse κ but lack human κ.

FIG. 51 shows the relative distribution of B cells expressing mouse λ onthe cell surface; 0011 mouse (7655) lymphocytes are positive for mouseλ; 0001 mouse (7656) lymphocytes do not express significant mouse λ;wildtype mouse (7777) lymphocytes express mouse λ but at a relativelylower level than the 0011 mouse (7655).

FIG. 52 shows the relative distribution of B cells positive forendogenous mouse λ as compared to human λ (transgene-encoded). The upperleft panel shows the results of cells from a wildtype mouse possessingfunctional endogenous heavy and light chain alleles and lacking humantransgene(s); the cells are positive for mouse lambda. The upper rightpanel shows cells from a mouse (#5822) having a κ knockout background(JKD++) and harboring the human κ transgene intergration of theKC1e-1399 line; the cells are positive for human κ or mouse λ in roughlyproportional amounts. The lower left panel shows cells from a mouse(#7132) having a κ knockout background (JKD++) and harboring the human κtransgene intergration of the KC2-1610 line; more cells are positive formouse λ than for human κ, possibly indicating that the KC2-1610transgene integration is less efficient than the KC1e-1399 transgeneintegration. The lower right panel shows cells from a mouse harboring ahuman κ minilocus transgene (KCo4) and lacking a functional endogenousmurine κ allele. The data presented in FIG. 52 also demonstrates thevariability of phenotypic expression between transgenes. Suchvariability indicates the desirability of selecting for individualtransgenes and/or transgenic lines which express one or more desiredphenotypic features resulting from the integrated transgene (e.g.,isotype switching, high level expression, low murine Ig background).Generally, single or multiple transgene species (e.g., pKC1e, pKC2,KCo4) are employed separately to form multiple individual transgeniclines differing by: (1) transgene, (2) site(s) of transgene integration,and/or (3) genetic background. Individual transgenic lines are examinedfor desired parameters, such as: (1) capability to mount an immuneresponse to a predetermined antigen, (2) frequency of isotype switchingwithin transgene-encoded constant regions and/or frequency oftrans-switching to endogenous (e.g., murine) Ig constant region genes,(3) expression level of transgene-encoded immunoglobulin chains andantibodies, (4) expression level of endogenous (e.g., murine)immunoglobulin immunoglobulin sequences, and (5) frequency of productiveVDJ and VJ rearrangement. Typically, the transgenic lines which producethe largest concentrations of transgene-encoded (e.g., human)immunoglobulin chains are selected; preferably, the selected linesproduce about at least 40 μg/ml of transgene-encoded heavy chain (e.g.,human μ or human γ) in the serum of the transgenic animal and/or aboutat least 100 μg/ml of transgene-encoded light chain (e.g., human κ).

Mice were examined for their expression of human and murineimmunoglobulin chains in their unimmunized serum and in their serumfollowing immunization with a specific antigen, human CD4. FIG. 53 showsthe relative expression of human μ, human γ, murine μ, murine γ, humanκ, murine κ, and murine λ chains present in the serum of four separateunimmunized 0011 mice of various genotypes (nt=not tested); human κpredominates as the most abundant light chain, and human μ and murine γ(putatively a product of trans-switching) are the most abundant heavychains, with variability between lines present, indicating the utilityof a selection step to identify advantageous genotypic combinations thatminimize expression of murine chains while allowing expression of humanchains. Mice #6907 and 7088 show isotype switching (cis-switching withinthe transgene) from human μ to human γ.

FIG. 54 shows serum immunoglobulin chain levels for human μ (huμ), humanγ (huγ), human κ (huκ), murine μ (msμ), murine γ (msγ), murine κ (msκ),and murine λ (msλ) in mice of the various 0011 genotypes.

Specific Antibody Response in 0011 Mice

An 0011 mouse (#6295) was immunized with an immunogenic dose of humanCD4 according to the following immunization schedule: Day 0,intraperitoneal injection of 100 μl of CD4 mouse immune serum; Day 1,inject 20 μg of human CD4 (American Bio-Tech) on latex beads with DDA in100 μl; Day 15 inject 20 μg of human CD4 (American Bio-Tech) on latexbeads with DDA in 100 μl; Day 29 inject 20 μg of human CD4 (AmericanBio-Tech) on latex beads with DDA in 100 μl; Day 43 inject 20 μg ofhuman CD4 (American Bio-Tech) on latex beads with DDA in 100 μl.

FIG. 55 shows the relative antibody response to CD4 immunization at 3weeks and 7 weeks demonstrating the presence of human μ, human κ, andhuman γ chains in the anti-CD4 response. Human γ chains are present atsignificantly increased abundance in the 7 week serum, indicating thatcis-switching within the heavy chain transgene (isotype switching) isoccurring in a temporal relationship similar to that of isotypeswitching in a wildtype animal.

FIG. 56 shows a schematic compilation of various human heavy chain andlight chain transgenes.

Example 28

This example provides for the targeted knockout of the murine λ lightchain locus.

Targeted Inactivation of the Murine Lambda Light Chain Locus

Unlike the Ig heavy and kappa light chain loci, the murine VλJλ and Cλgene segments are not grouped into 3 families arranged in a 5′ to 3′array, but instead are interspersed. The most 5′ portion consists of twoV segments (Vλ2 and VλX) which are followed, proceeding in a 3′direction, by two constant region exons, each associated with its own Jsegment (Jλ2Cλ2 and the pseudogene Jλ4Cλ4). Next is the most extensivelyused V segment (Vλ1) which is followed by the second cluster of constantregion exons (Jλ3Cλ3 and Jλ1Cλ1). Overall the locus spans approximate200 kb, with intervals of ˜20-90 kb between the two clusters.

Expression of the lambda locus involves rearrangement of Vλ2 or VλXpredominantly to Jλ2 and only rarely further 3′ to Jλ3 or Jλ1. Vλ1 canrecombine with both Jλ3 and Jλ1. Thus the lambda locus can be mutated inorder to fully eliminate recombination and expression of the locus.

The distance between the two lambda gene clusters makes it difficult toinactivate expression of the locus via the generation of a singlecompact targeted deletion, as was used in inactivating the murine Igheavy and kappa light chain loci. Instead, a small single deletion whichwould eliminate expression lambda light chains spans approximately 120kb, extending from Jλ2Cλ2 to Jλ1Cλ1 (FIG. 57). This removes all of thelambda constant region exons as well as the Vλ1 gene segment, ensuringinactivation of the locus.

Replacement type targeting vectors (Thomas and Capecchi (1987) op.cit)are constructed in which the deleted 120 kb is replaced with theselectable marker gene, neo, in a PGK expression cassette. The marker isembedded within genomic lambda sequences flanking the deletion toprovide homology to the lambda locus and can also contain the HSV-tkgene, at the end of one of the regions of homology, to allow forenrichment for cells which have homologously integrated the vectors.Lambda locus genomic clone sequences are obtained by screening of astrain 129/Sv genomic phage library isogenic to the ES line beingtargeted, since the use of targeting vectors isogenic to the chromosomalDNA being targeted has been reported to enhance the efficiency ofhomologous recombination. Targeting vectors are constructed which differin their lengths of homology to the lambda locus. The first vector(vector 1 in FIG. 58) contains the marker gene flanked by total ofapproximately 8-12 kb of lambda locus sequences. For targeting events inwhich replacement vectors mediate addition or detection of a few kb ofDNA this has been demonstrated to be a more than sufficient extent ofhomology (Hasty et al. (1991) op.cit; Thomas et al. (1992) op.cit.).Vectors with an additional approximately 40-60 kb of flanking lambdasequence are also constructed (vector 2 in FIG. 58). Human Ig minilociof at least 80 kb are routinely cloned and propagated in the plasmidvector pGP1 (Taylor et al. (1993) op.cit).

An alternative approach for inactivation of the lambda locus employs twoindependent mutations, for example mutations of the two constant regionclusters or of the two V region loci, in the same ES cell. Since bothconstant regions are each contained within ˜6 kb of DNA, whereas one ofthe V loci spans ˜19 kb, targeting vectors are constructed toindependently delete the Jλ2Cλ2/Jλ4Cλ4 and the Jλ3Cλ3/Jλ1Cλ1 loci. Asshown in FIG. 58, each vector consists of a selectable marker (e.g., neoor pac) in a PGK expression cassette, surrounded by a total of ˜8-12 kbof lambda locus genomic DNA blanking each deletion. The HSV-tk gene canbe added to the targeting vectors to enrich for homologous recombinationevents by positive-negative selection. ES cells are targetedsequentially with the two vectors, such that clones are generated whichcarry a deletion of one of the constant region loci; these clones arethen targeted sequentially with the two vectors, such that clones willbe generated which carry a deletion of one of the constant region loci,and these clones are then targeted to generate a deletion of theremaining functional constant region cluster. Since both targetingevents are thus being directed to the same cell, it is preferable to usea different selectable marker for the two targetings. In the schematicexample shown in FIG. 58, one of the vectors contains the neo gene andthe other the pac (puromycin N-acetyl transferase) gene. A thirdpotential dominant selectable marker is the hyg (hygromycinphosphotransferase) gene. Both the pac and hyg genes can be beeninserted into the PGK expression construct successfully used fortargeting the neo gene into the Ig heavy and kappa light chain loci.Since the two lambda constant region clusters are tightly linked, it isimportant that the two mutations reside on the same chromosome. Therepreferably is a 50% probability of mutating the same allele by twoindependent targeting events, and linkage of the mutations isestablished by their co-segregation during breeding of chimeras derivedfrom the doubly targeted ES cells.

Example 29

This example provides for the targeted knockout of the murine heavychain locus.

Targeted Inactivation of the Murine Heavy Chain Locus

A homologous recombination gene targeting transgene having the structureshown in FIG. 59 is used to delete at least one and preferablysubstantially all of the murine heavy chain locus constant region genesby gene targeting in ES cells. FIG. 59 shows a general schematic diagramof a targeting transgene. Segment (a) is a cloned genomic DNA sequencelocated upstream of the constant region gene(s) to be deleted (i.e,proximal to the J_(H) genes); segment (b) comprises a positive selectionmarker, such as pgk-neo; segment (c) is a cloned genomic DNA sequencelocated downstream of the constant region gene(s) to be deleted (i.e,distal to the constan region gene(s) and J_(H) genes); and segment (d),which is optional, comprises a negative selection marker gene (e.g.,HSV-tk). FIG. 60 shows a map of the murine heavy chain locus as takenfrom Immunoglobulin Genes, Honjo, T, Alt, F W, and Rabbits T H (eds.)Academic Press, NY (1989) p. 129.

A targeting transgene having a structure according to FIG. 59, wherein:(1) the (a) segment is the 11.5 kb insert of clone JH8.1 (Chen et al.(1993) Int. Immunol. 5: 647) or an equivalent portion comprising aboutat least 1-4 kb of sequence located upstream of the murine Cμ gene, (2)the (b) segment is pgk-neo as described supra, (3) the (c) segmentcomprises the 1674 bp sequence shown in FIG. 61 or a 4-6 kb insertisolated from a phage clone of the mouse Cα gene isolated by screening amouse genomic clone library with the end-labeled oligonucleotide havingthe sequence: 5′-gtg ttg cgt gta tca gct gaa acc tgg aaa cag ggt gaccag-3′ (SEQ ID NO.: 418) and (4) the (d) segment comprises the HSV-tkexpression cassette described supra.

Alternatively, a stepwise deletion of one or more heavy chain constantregion genes is performed wherein a first targeting transgene compriseshomology regions, i.e., segments (a) and (c), homologous to sequencesflanking a constant region gene or genes, a first species of positiveselection marker gene (pgk-neo), and an HSV-tk negative selectionmarker. Thus, the (a) segment can comprise a sequence of at least about1-4 kb and homologous to a region located upstream of Cγ3 and the (c)segment can comprise a sequence of at least about 1-4 kb and homologousto a region located upstream of Cγ2a. This targeting transgene deletesthe Cγ³, Cγ1, Cγ72b, and Cγ2a genes. This first targeting transgene isintroduced into ES cells and correctly targeted recombinants areselected (e.g., with G418), producing a correctly targeted C regiondeletion. Negative selection for loss of the HSV-tk cassette is thenperformed (e.g., with ganciclovir or FIAU). The resultant correctlytargeted first round C deletion recombinants have a heavy chain locuslacking the Cγ3, Cγ1, Cγ2b, and Cγ2a genes.

A second targeting transgene comprises homology regions, i.e., segments(a) and (c), homologous to sequences flanking a constant region gene orgenes, a second species of positive selection marker gene different thatthe first species (e.g., gpt or pac), and an HSV-tk negative selectionmarker. Thus, the (a) segment can comprise a sequence of at least about1-4 kb and homologous to a region located upstream of Cε and the (c)segment can comprise a sequence of at least about 1-4 kb and homologousto a region located upstream of Cα. This targeting transgene deletes theCε and Cα genes.

This second targeting transgene is introduced into the correctlytargeted C-region recombinant ES cells obtained from the first targetingevent. Cells which are correctly targeted for the second knockout event(i.e., by homologous recombination with the second targeting transgene)are selected for with a selection drug that is specific for the secondspecies of positive selection marker gene (e.g., mycophenolic acid toselect for gpt; puromycin to select for pac). Negative selection forloss of the HSV-tk cassette is then performed (e.g., with ganciclovir orFIAU). These resultant correctly targeted second round C regionrecombinants have a heavy chain locus lacking the Cγ3, Cγ1, Cγ2b, Cγ2a,Cε, and Cα genes.

Correctly targeted first-round or second-round recombinant ES cellslacking one or more C region genes are used for blastocyst injections asdescribed (supra) and chimeric mice are produced. Germline transmissionof the targeted heavy chain alleles is established, and breeding of theresultant founder mice is performed to generate mice homozygous forC-region knockouts. Such C-region knockout mice have several advantagesas compared to J_(H) knockout mice; for one example, C-region knockoutmice have diminished ability (or completely lack the ability) to undergotrans-switching between a human heavy chain transgene and an endogenousheavy chain locus constant region, thus reducing the frequency ofchimeric human/mouse heavy chains in the transgenic mouse. Knockout ofthe murine gamma genes is preferred, although μ and delta are frequentlyalso deleted by homologous targeting. C-region knockout can be done inconjunction with other targeted lesions in the endogenous murine heavychain locus; a C-region deletion can be combined with a J_(H) knockoutto preclude productive VDJ rearrangement of the murine heavy chain locusand to preclude or reduce trans-switching between a human heavy chaintransgene and the murine heavy chain locus, among others. For someembodiments, it may be desirable to produce mice which specifically lackone or more C-region genes of the endogenous heavy chain locus, butwhich retain certain other C-region genes; for example, it may bepreferable to retain the murine Cα gene to allow to production ofchimeric human/mouse IgA by trans-switching, if such IgA confers anadvantageous phenotype and does not substantially interfere with thedesired utility of the mice.

Example 30

This example demonstrates ex vivo depletion of lymphocytes expressing anendogenous (murine) immunoglobulin from a lymphocyte sample obtainedfrom a transgenic mouse harboring a human transgene. The lymphocytesexpressing murine Ig are selectively depleted by specific binding to ananti-murine immunoglobulin antibody that lacks substantial binding tohuman immunoglobulins encoded by the transgene(s).

Ex Vivo Depletion of Murine Ig-Expressing B-cells

A mouse homozygous for a human heavy chain minilocus transgene (HC2) anda human light chain minilocus transgene (KCo4) is bred with a C57BL/6(B6) inbred mouse-to obtain 2211 mice (i.e., mice which: are homozygousfor a functional endogenous murine heavy chain locus, are homozygous fora functional endogenous murine light chain locus, and which possess onecopy of a human heavy chain transgene and one copy of a human lightchain transgene). Such 2211 mice also express B6 major and minorhistocompatibility antigens. These mice are primed with an immunogenicdose of an antigen, and after approximately one week spleen cells areisolated. B cells positive for murine Ig are removed by solidphase-coupled antibody-dependent cell separation according to standardmethods (Wysocki et al. (1978) Proc. Natl. Acad. Sci. (U.S.A.) 75: 2844;MACS magnetic cell sorting, Miltenyi Biotec Inc., Sunnyvale, Calif.),followed by antibody-dependent complement-mediated cell lysis (SelectedMethods in Cellular Immunology, Mishell B B and Shiigi S M (eds.), W.H.Freeman and Company, New York, 1980, pp. 211-212) to substantiallyremove residual cells positive for murine Ig. The remaining cells in thedepleted sample (e.g., T cells, B cells positive for human Ig) areinjected i.v., preferably together with additional anti-murine Igantibody to deplete arising B cells, into a SCID/B6 or RAG/B6 mouse. Thereconstitutued mouse is then further immunized for the antigen to obtainantibody and affinity matured cells for producing hybridoma clones.

Example 31 Production of Fully Human Antibodies in Somatic Chimeras

A method is described for producing fully human antibodies in somaticchimeric mice. These mice are generated by introduction of embryonicstem (ES) cells, carrying human immunoglobulin (Ig) heavy and lightchain transgenes and lacking functional murine Ig heavy and kappa lightchain genes, into blastocysts from RAG-1 or RAG-2 deficient mice.

RAG-1 and RAG-2 deficient mice (Mombaerts et al. (1992) Cell 68: 869;Shinkai et al. (1992) Cell 68: 855) lack murine B and T cells due to aninability to initiate VDJ rearrangement and to assemble the genesegments encoding Igs and T cell receptors (TCR). This defect in B and Tcell production can be complemented by injection of wild-type ES cellsinto blastocysts derived from RAG-2 deficient animals. The resultingchimeric mice produce mature B and T cells derived entirely from theinjected ES cells (Chen et al. (1993) Proc. Natl. Acad. Sci. USA 90:4528).

Genetic manipulation of the injected ES cells is used for introducingdefined mutations and/or exogenous DNA constructs into all of the Band/or T cells of the chimeras. Chen et al. (1993), Proc. Natl. Acad.Sci. USA 90:4528-4532) generated ES cells carrying a homozygousinactivation of the Ig heavy chain locus, which, when injected into RAGblastocysts, produced chimeras which made T cells in the absence of Bcells. Transfection of a rearranged murine heavy chain into the mutantES cells results in the rescue of B cell development and the productionof both B and T cells in the chimeras.

Chimeric mice which express fully human antibodies in the absence ofmurine Ig heavy chain or kappa light chain synthesis can be generated.Human Ig heavy and light chain constructs are introduced into ES cellshomozygous for inactivation of both the murine Ig heavy and kappa lightchain genes. The ES cells are then injected into blastocysts derivedfrom RAG2 deficient mice. The resulting chimeras contain B cells derivedexclusively from the injected ES cells which are incapable of expressingmurine Ig heavy and kappa light chain genes but do express human Iggenes.

Generation of ES Cells Homozygous for Inactivation of the ImmunoglobulinHeavy and Kappa Light Chain Genes

Mice bearing inactivated Ig heavy and kappa light chain loci weregenerated by targeted deletion, in ES cells, of Ig J_(H) and J_(K)/C_(K)sequences, respectively according to known procedures (Chen et al.(1993) EMBO J. 12: 821; and Chen et al. (1993) Int. Immunol. op.cit).The two mutant strains of mice were bred together to generate a strainhomozygous for inactivation of both Ig loci. This double mutant strainwas used for derivation of ES cells. The protocol used was essentiallythat described by Robertson (1987, in Teratocarcinomas and EmbryonicStem Cells: A Practical Approach, p. 71-112, edited by E. J. Robertson,IRL Press). Briefly, blastocysts were generated by natural matings ofhomozygous double mutant mice. Pregnant females were ovariectomized onday 2.5 of gestation and the “delayed” blastocysts were flushed from theuterus on day 7 of gestation and cultured on feeder cells, to helpmaintain their undifferentiated state. Stem cells from the inner cellmass of the blastocysts, identifiable by their morphology, were picked,dissociated, and passaged on feeder cells. Cells with a normal karyotypewere identified, and male cell lines will be tested for their ability togenerate chimeras and contribute to the germ cells of the mouse. Male EScells are preferable to female lines since a male chimera can producesignificantly more offspring.

Introduction of Human Ig Genes into Mouse Ig Heavy and Kappa Light ChainDeficient ES Cells

Human immunoglobulin heavy and light chain genes are introduced into themutant ES cells as either minilocus constructs, such as HC2 and KC-C04,or as YAC clones, such as J1.3P. Transfection of ES cells with human IgDNAs is carried out by techniques such as electroporation or lipofectionwith a cationic lipid. In order to allow for selection of ES cells whichhave incorporated the human DNA, a selectable marker either is ligatedto the constructs or is co-transfected with the constructs into EScells. Since the mutant ES cells contain the neomycin phosphotransferse(neo) gene as a result of the gene targeting events which generated theIg gene inactivations, different selectable markers, such as hygromycinphosphotransferase (hyg) or puromycin N-acetyl transferase (pac), areused to introduce the human Ig genes into the ES cells.

The human Ig heavy and light chain genes can be introducedsimultaneously or sequentially, using different selectable markers, intothe mutant ES cells. Following transfection, cells are selected with theappropriate selectable marker and drug-resistant colonies are expandedfor freezing and for DNA analysis to verify and analyze the integrationof the human gene sequences.

Generation of Chimeras

ES clones containing human Ig heavy and light chain genes are injectedinto RAG-2 blastocysts as described (Bradley, A. (1987), inTeratocarcinomas and Embryonic Stem Cells: A Practical Approach, p.113-151, edited by E. J. Robertson, IRL Press) and transferred into theuteri of pseudopregnant females. Offspring are screened for the presenceof human antibodies by ELISA assay of serum samples. Positive animalsare used for immunization and the production of human monoclonalantibodies.

Example 32

This example describes the introduction, via homologous recombination inES cells, of a targeted frameshift mutation into the murine heavy chainlocus leading to a deletion of B cells which undergo switchrecombination. The frameshifted mice are suitable hosts for harboringnon-murine (e.g., human) transgenes encoding human sequenceimmunoglobulins.

The novel frameshifted mice can be used for expressing non-murine (e.g.,human) sequence immunoglobulins encoded by heavy chain transgene(s)and/or light chain transgene(s), and for the isolation of hybridomasexpressing class-switched, affinity matured, human sequence antibodiesfrom introduced transgenes, among other uses. A frameshift is introducedinto one of the four mouse JH gene segments and into the first exon ofthe mouse μ gene. The two introduced frameshift mutations compensate foreach other thus allowing for the expression of fully functional murine μheavy chain when a B cell uses the frameshifted JH for a functional VDJjoint. None of the other three JH segments can be used for functionalVDJ joining because of the frameshift in μ, which is not compensated inthe remaining JH genes. Alternatively, compensating frameshifts can beengineered into multiple murine JH genes.

A mouse homozygous for a compensated, frameshifted immunoglobulin heavychain allele has an approximately physiological level of peripheral Bcells, and an approximately physiological level of serum IgM comprisingboth murine and human μ. However, B cells recruited into germinalcenters frequently undergo a class switch to a non-μ isotype. Such aclass switch in B cells expressing the endogenous murine μ chain leadsto the expression of a non-compensated frameshift mRNA, since theremaining non-μ C_(H) genes do not possess a compensating frameshift.The resulting B cells do not express a B cell receptor and are deleted.Hence, B cells expressing a murine heavy chain are deleted once theyreach the stage of differentiation where isotype switching occurs.However, B cells expressing heavy chains encoded by a non-murine (e.g.,human) transgene capable of isotype switching and which does not containsuch isotype-restrictive frameshifts are capable of further development,including isotype switching and/or affinity maturation, and the like.

Therefore, the frameshifted mouse has an impaired secondary responsewith regard to murine heavy chain (μ) but a significant secondaryresponse with regard to transgene-encoded heavy chains. If a heavy chaintransgene that is capable of undergoing class switching is introducedinto this mutant background, the non-IgM secondary response is dominatedby transgene expressing B cells. It is thus possible to isolate affinitymatured human sequence immunoglobulin expressing hybridomas from theseframeshifted mice. Moreover, the frameshifted mice generally possessimmunoprotective levels of murine IgM, which may be advantageous wherethe human heavy chain transgene can encode only a limited repertoire ofvariable regions.

For making hybridomas secreting human sequence monoclonal antibodies,transgenic mutant mice are immunized; their spleens fused with a myelomacell line; and the resulting hybridomas screened for expression of thetransgene encoded human non-μ isotype. Further, the frameshifted mousemay be advantageous over a JH deleted mouse because it will contain afunctional μ switch sequence adjacent to a transcribed VDJ which servesas an active substrate for cis-switching (Gu et al. (1993) Cell 73:1155); thus reducing the level of trans-switched B cells that expresschimeric human/mouse antibodies.

Construction of Frameshift Vectors

Two separate frameshift vectors are built. One of the vectors is used tointroduce 2 nucleotides at the 3′ end of the mouse J4 gene segment, andone of the vectors is used to delete those same two nucleotides from the5′ end of exon 1 of the mouse μ gene.

1. JH Vector.

A 3.4 kb XhoI/EcoRI fragment covering the mouse heavy chain J region andthe μ intronic enhancer is subcloned into a plasmid vector that containsa neomycin resistance gene as well as a herpes thymidine kinase geneunder the control of a phosphoglycerate kinase promoter (tk/neocassette; Hasty et al., (1991) Nature 350: 243). This clone is then usedas a substrate for generating 2 different PCR fragments using thefollowing oligonucleotide primers:

o-A1 (SEQ ID NO. 161) 5′-cca cac tct gca tgc tgc aga agc ttt tct gta-3′o-A2 (SEQ ID NO. 162) 5′-ggt gac tga ggt acc ttg acc cca gta gtc cag-3′o-A3 (SEQ ID NO. 163) 5′-ggt tac ctc agt cac cgt ctc ctc aga ggt aag aatggc ctc-3′ o-A4 (SEQ ID NO. 164) 5′-agg ctc cac cag acc tct cta gac agcaac tac-3′

Oligonucleotides o-A1 and o-A2 are used to amplify a 1.2 kb fragmentwhich is digested with SphI and KpnI. Oligonucleotides o-A3 and o-A4 areused to amplify a 0.6 kb fragment which is digested with KpnI and XbaI.These two digested fragments are then cloned into SphI/XbaI digestedplasmid A to produce plasmid B.

Plasmid B contains the 2 nucleotide insertion at the end of the J4 and,in addition, contains a new KpnI site upstream of the insertion. TheKpnI site is used as a diagnostic marker for the insertion.

Additional flanking sequences may be cloned into the 5′ XhoI site andthe 3′ EcoRI site of plasmid B to increase its homologous recombinationefficiency. The resulting plasmid is then digested with SphI, or anotherrestriction enzyme with a single site within the insert, andelectroporated into embryonic stem cells which are then selected withG418 as described by Hasty et al. (1991) op.cit. Homologous recombinantsare identified by Southern blot hybridization and then selected withFIAU as described by Hasty et al. to obtain deleted subclones whichcontain only the 2 base pair insertion and the new KpnI site in JH4.These are identified by Southern blot hybridization of KpnI digested DNAand confirmed by DNA sequence analysis of PCR amplified JH4 DNA.

The resulting mouse contains a JH4 segment that has been converted fromthe unmutated sequence:

. . . TGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAG_gtaagaatggcctctcc . . . SEQ IDNO. (165)       TrpGlyGlnGlyThrSerValThrValSerSerGlu SEQ ID NO. (166)

to the mutant sequence:

. . . TGGGGTCAAGGTACCTCAGTCACCGTCTCCTCAGAGgtaagaatggcctctcc . . . SEQ IDNO. (167)        TrpGlyGlnGlyThrSerValThrValSerSerGlu SEQ ID NO. (168)μ Exon 1 Vector

Using similar in vitro mutagenesis methodology described above toengineer a two base pair insertion into the JH4 gene segment, PCRproducts and genomic subclones are assembled to create a vectorcontaining a two base pair deletion at the 5′ end of the first μ exon.In addition, to mark the mutation, a new XmnI site is also introduceddownstream by changing an A to a G.

The sequence of the unmutated μ gene is:

. . . ctggtcctcagAGAGTCAGTCCTTCCCAAATGTCTTCCCCCTCGTC . . . SEQ ID NO.(169)                 GluSerGlnSerPheProAsnValpheProLeuVal SEQ ID NO.(170)

The sequence of the mutated μ gene is:

                                XmnI . . .ctggtcctcag_AGTCAGTCCTTCCCGAATGTCTTCCCCCTCGTC . . . (SEQ ID NO.171)                  SerGlnSerPheProAsnValPheProLeuVal (SEQ ID NO.172)The homologous recombination vector containing the mutant sequence islinearized and electroporated into an ES cell line containing the JH4insertion. Homologous recombinants are identified fromneomycin-resistant clones. Those homologous recombinants that containthe frameshift insertion on the same chromosome as the JH4 insertion areidentified by Southern blot hybridization of KpnI/BamHI digested DNA.The JH4 insertion is associated with a new KpnI site that reduces thesize of the J-μ intron containing KpnI/BamHI fragment from the wild type11.3 kb to a mutant 9 kb. The resulting clones are then selected fordeletion of the inserted tk/neo cassette using FIAU. Clones containingthe mutant μ exon are identified by Southern blot hybridization of XmnIdigested DNA. The mutation is confirmed by DNA sequence analysis of PCRamplified μ exon1 DNA.Generation of Frameshifted Mice

The ES cell line containing both the two base pair insertion in JH4, andthe two base pair deletion in μ exon 1, is then introduced intoblastocyst stage embryos which are inserted into pseudopregnant femalesto generate chimeras. Chimeric animals are bred to obtain germlinetransmission, and the resulting animals are bred to homozygosity toobtain mutant animals homozygous for compensated frameshifted heavychain loci and having impaired secondary humoral immune responses in Bcells expressing murine heavy chains.

A human heavy chain transgene, such as for example pHC1 or pHC2 and thelike, may be bred into the murine heavy chain frameshift background bycrossbreeding mice harboring such a human transgene into mice having theframeshifted murine IgH locus. Via interbreeding and backcrossing, micehomozygous at the murine IgH locus for μ-compensated frameshifted murineIgH alleles (i.e., capable of compensated in-frame expression of onlymurine μ and not murine non-μ chains) and harboring at least oneintegrated copy of a functional human heavy chain transgene (e.g., pHC1or pHC2) are produced. Such mice may optionally contain knockout ofendogenous murine κ and/or λ loci as described supra, and may optionallycomprise a human or other non-murine light chain transgene (e.g., pKC1e,pKC2, and the like).

Alternatively, the human transgene(s) (heavy and/or light) may comprisecompensating frameshifts, so that the transgene J gene(s) contain aframeshift that is compensated by a frameshift in the transgene constantregion gene(s). Trans-switching to the endogenous constant region genesis uncompensated and produces a truncated or nonsense product; B cellsexpressing such uncompensated trans-switched immunoglobulins areselected against and depleted.

Example 33 Endogenous Heavy Chain Inactivation by D Region Ablation

This example describes a positive-negative selection homologousrecombination vector for replacing the mouse germline immunoglobulinheavy chain D region with a nonfunctional rearranged VDJ segment. Theresulting allele functions within a B cell as a normal non-productiveallele, with the allele undergoing intra-allele heavy chain classswitching, thereby reducing the level of trans-switching to an activetransgene locus.

D Region Targeting Construct

An 8-15 kb DNA fragment located upstream of the murine D region isisolated and subcloned from a mouse strain 129 phage library using anoligonucleotide probe comprising approximately 50 consecutivenucleotides of the published sequence for the DFL16.1 segment listed inGenBank. DFL16.1 is the upstream D segment (i.e., proximal to the Vregion gene cluster and distal to the constant region gene cluster).

Similarly, a 9.5 kb BamHI fragment containing JH3, JH4, Eμ, Sμ, and thefirst two coding exons of the μ constant region is isolated andsubcloned from a mouse strain 129 genomic phage library.

A 5-10 kb rearranged VDJ is then isolated from a mouse hybridoma (anystrain) and a synthetic linker containing a stop codon is inserted intothe J segment. The stop linker within the J is preferable to anout-of-frame VDJ junction because of the possibility of V replacementrearrangements.

These three fragments are assembled together with a PGKneo positiveselection cassette and a PGKHSVtk negative selection cassette to form apositive-negative selection vector for eliminating the mouse D region in129-derived ES cells (e.g., AB1) by homologous recombination. Thetargeting vector is formed by ligating the 8-15 kb DNA fragment to thepositive selection cassette (e.g., PGKneo), which is itself ligated tothe rearranged 5-10 kb rearranged VDJ, which is itself ligated to the9.5 kb BamHI fragment; the negative selection cassette (e.g., PGKHSVtk)is then ligated at either end of the targeting construct. Theconstruction of such a D region targeting vector is shown schematicallyin FIG. 63.

The D region targeting construct is transferred into AB1 ES cells,positive and negative selection is performed as described above, andcorrectly targeted ES cells are cloned. The correctly targeted ES cellclones are used for blastocyst injections and chimeric mice areproduced. The chimeric mice are bred to produce founder mice harboring aD-region inactivated heavy chain allele. Interbreeding of offspring isperformed to produce homozygotes lacking a functional endogenous heavychain locus. Such homozygotes are used to crossbreed to mice harboringhuman Ig transgenes (e.g., pHC1, pHC2, pKC2, pKC1e, KCo4) to yield (byfurther backcrossing to the homozygotes lacking a functional D-region)mice lacking a functional endogenous heavy chain locus and harboring ahuman heavy transgene (and preferably also a human light chaintransgene). In embodiments where some functional endogenous light chainloci remain (e.g., λ loci), it is generally preferred that transgenescontain transcriptional control sequences that direct high levelexpression of human light chain (e.g., κ) polypeptides, and thus allowthe transgene locus to compete effectively with the remaining endogenouslight chain (e.g., λ) loci. For example, the Co4 kappa light chaintransgene is generally preferred as compared to pKC1 with regard to theability to compete effectively with the endogenous λ loci in thetransgenic animal.

Example 34

This example describes expansion of the human light chain transgene Vgene repertoire by co-injection of a human κ light chain minilocus and ayeast artificial chromosome comprising a portion of the human Vκ locus.

Introduction of Functional Human Light Chain V Segments by Co-Injectionof Vκ-Containing YAC DNA and a κ Minilocus

An approximately 450 kb YAC clone containing part of the human Vκ locuswas obtained as a non-amplified YAC DNA from clone 4×17E1 of thepublicly available ICRF YAC library (Larin et al. (1991) Proc. Natl.Acad. Sci. (U.S.A.) 88: 4123; Genome Analysis Laboratory, ImperialCancer Research Fund, London, UK). The 450 kb YAC clone was isolatedwithout prior amplification by standard pulsed-field gel electrophoresisas per the manufacturer's specifications (CHEF DR-II electrophoresiscell, Bio-Rad Laboratories, Richmond, Calif.). Six individual pulsefield gels were stained with ethidium bromide and the gel materialcontaining the YAC clone DNA was excised from the gel and then embeddedin a new (low melting point agarose in standard gel buffer) gel cast ina triangular gel tray. The resulting triangular gel (containing the sixexcised YAC-containing gel blocks) was extended at the apex with anarrow agarose gel with 2 M NaOAc in addition to the standardelectrophoresis buffer. The gel was then placed in an electrophoresischamber immersed in standard gel buffer. The Y-shaped gel former risesabove the surface of the buffer so that current can only flow to thenarrow high salt gel portion. A plexiglass block was placed over thehigh salt gel slice to prevent diffusion of the NaOAc into the buffer.The YAC DNA was then electrophoresed out of the original excised gelsliced (embedded) and into the narrow high salt gel portion. At thepoint of transition from the low salt gel to the high salt gel, there isa resistance drop that effectively halts the migration of the DNA at theapex of the triangular gel.

Following electrophoresis and staining with ethidium bromide, theconcentrated YAC DNA was cut away from the rest of the gel and theagarose was digested with GELase (EpiCentre Technologies, Madison,Wis.). Cesium chloride was then added to the resultant YAC-containingliquid to obtain a density of 1.68 g/ml. This solution was centrifugedat 37,000 rpm for 36 hours to separate the YAC DNA from anycontaminating material. 0.5 ml fractions of the resulting densitygradient were isolated and the peak DNA fraction was dialyzed against 5mM Tris (pH 7.4), 5 mM NaCl, 0.1 M EDTA. Following dialysis, theconcentration of the resulting 0.65 ml solution of YAC DNA was found tocontain 2 μg/ml of DNA. This YAC DNA was mixed with purified DNA insertfrom plasmids pKC1B and pKV4 at a ratio of 20:1:1 (microgramsYAC4x17E1:KC1B:KV4). The resulting 2 μg/ml solution was injected intothe pronuclei of half-day B6CBF2 embryos, and 95 surviving microinjectedembryos were transferred into the oviducts of pseudopregnant females.Twelve mice which developed from the microinjected embryos were born.

Example 35

This example describes class-switching, somatic mutation, and B celldevelopment in immunized transgenic mice homozygous for an inactivatedendogenous immunoglobulin locus and containing the HC1 or HC2 heavychain transgene(s).

To demonstrate that a human sequence germline configuration minilocuscan functionally replace the authentic locus, we bred a mouse strainlacking endogenous IgH with strains containing humangermline-configuration IgH transgenes. The two transgene miniloci, HC1and HC2, include one and four functional variable (V) segmentsrespectively 10 and 16 diversity (D) segments respectively, all sixjoining (JH) segments, and both the μ and γ1 constant region segments.The miniloci include human cis-acting regulatory sequences—such as theJH-μ intronic enhancer and the μ and γ1 switch sequences—that areclosely linked to the coding segments. They also include an additionalenhancer element derived from the 3′ end of the rat IgH locus. Wecrossed HC1 and HC2 transgenic mice with stem-cell derived mutant micethat lack JH segments (JHD mice) as described (supra) and cannottherefore undergo functional heavy chain rearrangements. The resultingtransgenic-JHD mice contain B cells that are dependent on the introducedheavy chain sequences.

Immunizations and Hybridomas.

We immunized mice by intraperitoneal injections of 50-100 μg of antigen.Antigens included human carcinoembryonic antigen (CEA; Crystal Chem,chicago, Ill.), hen eggwhite lysozyme (HEL; Pierce, Rockford, Ill.), andkeyhole limpet hemocyanin (KLH; Pierce, Rockford, Ill.). For primaryinjections we mixed the antigen with complete Freund's adjuvant, forsubsequent injections we used incomplete Freund's adjuvant (Gibco BRL,Gaithersburg, Md.). We fused spleen cells with the non-secreting mousemyeloma P3×63-Ag8.653 (ATCC, CRL1580). We assayed serum samples andhybridoma supernatants for the presence of specific and non-specificantibody comprising human heavy chain sequences by ELISA. For detectionof non-specific antibodies we coated microtiter wells with human heavychain isotype specific antibody (mouse MAb α human IgG1, clone HP6069,Calbiochem, La Jolla, Calif.; mouse MAb a human IgM, clone CH6, TheBinding Site, Birmingham, UK) and developed with peroxidase conjugatedantisera (horseradish peroxidase conjugated affinity purified fabfragment from polyclonal goat a human IgG(fc), cat #109-036-098;affinity purified horseradish peroxidase conjugated polyclonal rabbit ahuman IgM(fc), cat #309-035-095. Jackson Immuno Research, West Grove,Pa.). For detection of antigen-specific antibodies we coated microtiterwells with antigen and developed with peroxidase-conjugated human heavychain isotype specific antisera. We detected bound peroxidase byincubation with hydrogen peroxide and2,2′-Azino-bis-(3-Ethylbenzthiazoline-6-Sulfonic Acid, Sigma Chem. Co.,St. Louis, Mo.). The reaction product is measured by absorption at 415nm, and corrected for absorption at 490 nm.

Flow Cytometry.

We prepared single cell suspensions from spleen, bone marrow, andperitoneal cavity, and lysed red cells with NH₄Cl, as described byMishell and Shiigi. The lymphocytes are stained with the followingreagents: Phycoerythrin conjugated anti-mouse IgK (clone X36; BectonDickinson, San Jose, Calif.), FITC conjugated anti-mouse IgD (clone SBA1, Southern Biotech, AL), FITC conjugated anti-mouse CD5 (clone 53-7.3;Becton Dickinson, San Jose, Calif.), FITC conjugated anti-mouse Igλ(clone R26-46; Pharmingen, San Diego, Calif.), and Cy-Chrome conjugatedanti-mouse B220 (clone RA3-6B2; Pharmingen, San Diego, Calif.). Weanalyzed the stained cells using a FACScan flow cytometer and LYSIS IIsoftware (Becton Dickinson, San Jose, Calif.). Most macrophages,neutrophils, and residual red cells are excluded by gating on forwardand side scatter.

Rescue of B Cell Compartment

In the peritoneal cavity of HC1 transgenic-JHD animals we find normallevels of CD5⁺ B cells and approximately one-quarter the normal level ofconventional CD5⁻ B cells. The transgenic peritoneal CD5⁺ B cells aresimilar to the so-called B-1 cells described in normal animals: they arelarger than conventional B and T lymphocytes, they express lower levelsof B220 than the conventional B cells found in the spleen, and theyinclude a higher proportion of λ light chain expressing cells. Over 90%of the splenic B cells express κ, while up to 50% of the peritoneal Bcells express λ. Thus, while the level of conventional B cells isuniformly reduced in all tissues, the level of B-1, which are reportedto have a much greater capacity for self-renewal, appears to be normalin the HC1 transgenic-JHD animals.

Class Switching.

In transgenic-JHD mice, repeated exposure to antigen results in theproduction of human γ1 antibodies as well as μ antibodies. We injectedhuman CEA into transgenic-JHD mice at weekly intervals and monitored theserum levels of antigen-specific IgM and IgG1 over a period of fourweeks (FIG. 63). At one week there is a detectable IgM response but noIgG1 response. However, the IgG1 response is greater than the IgMresponse after two weeks, and it continues to increase while the IgMresponse remains relatively constant. This pattern—an initial IgMreaction followed by an IgG reaction—is typical of a secondary immuneresponse; and it suggests that cis-acting sequences included in thetransgene may be responding to cytokines that direct class switching. Wehave considered three possible mechanisms for expression of non-μisotypes, each of which have been discussed in the literature. Thesemechanisms are: alternative splicing, which does not involve deletion ofthe μ gene; “δ-type” switching, which involved deletion of the μ genevia homologous recombination between flanking repeat sequences; andnon-homologous recombination between switch regions. The results of ourexperiments, described below, are indicative of a switch regionrecombination model.

Two types of non-deletional alternative splicing mechanisms can beinvoked to explain an isotype shift. First, it is possible that a singletranscript covering both μ and γ1 is expressed from the transgene; thistranscript could be alternatively spliced in response to cytokinesinduced by exposure to antigen. Alternative, a cytokine induced steriletranscript initiating upstream of γ1 could be trans-spliced to the μtranscript. If either of these mechanisms were responsible for theexpression of human γ1 sequences, then we would expect to be able toisolate hybridomas that express both μ and γ1. However, although we havescreened several hundred hybridomas expressing either human μ or humanγ1, we have not found any such double producer (μ⁺, γ1⁺) hybridomas.This indicates that expression of γ1 is accompanied by deletion of the μgene.

Deletion of the μ gene can be mediated by non-homologous recombinationbetween the μ and γ1 switch regions, or by homologous recombinationbetween the two flanking 400 bp direct repeats (Σμ and Σμ) that areincluded in the HC1 and HC2 transgenes. Deletional recombination between.rho.μ and Σμ has been reported to be responsible for the IgD⁺, IgM⁻phenotype of some human B cells. While the first mechanism,non-homologous switch recombination, should generate switch products ofvarying lengths, the second mechanism, Σμ/Σμ recombination, shouldalways generate the same product. We performed a Southern blot analysisof genomic DNA isolated from three hybridomas (FIG. 64A), one expressingμ and two expressing γ1. We find genomic rearrangements upstream of thetransgene γ1 only in the two the γ1 switch regions (FIG. 64B).Furthermore, neither of the observed structures is compatible withhomologous recombination between Σμ and Σμ. Our results are thereforeconsistent with a model for γ1 isotype expression mediated by deletionalnon-homologous recombination between the transgene encoded μ and γ1switch regions.

Trans-Switching.

In addition to human γ1, we find mouse γ in the serum of HC1 and HC2transgenic-JHD mice. We have also obtained mouse γ expressing hybridomasfrom these animals. Because the non-transgenic homozygous JHD animals donot express detectable levels of mouse immunoglobulins, we attribute theexpression of mouse γ in the HC1 and HC2 transgenic-JHD animals to thephenomenon of trans-switching. All of the transgenic hybridomas that wehave analyzed express either mouse or human constant region sequences,but not both. It is therefore unlikely that a trans-splicing mechanismis involved. We used PCR amplification to isolate cDNA clones oftrans-switch products, and determined the nucleotide sequence of 10 ofthe resulting clones (FIG. 65). The 5′ oligonucleotide in the PCRamplification is specific for the transgene encoded VH251, and the 3′oligonucleotide is specific for mouse γ1, γ2b, and γ3 sequences. We findexamples of trans-switch products incorporating all three of these mouseconstant regions.

Somatic Mutation.

Approximately 1% of the nucleotides within the variable regions of thetrans-switch products shown in FIG. 7 are not germline encoded. This ispresumably due to somatic mutation. Because the mutated sequence hasbeen translocated to the endogenous locus, the cis-acting sequencesdirecting these mutations could be located anywhere 3′ of the mouse γswitch. However, as we discuss below, we also observe somatic mutationin VDJ segments that have not undergone such translocations; and thisresult indicates that sequences required by heavy chain somatic mutationare included in the transgene.

To determine if the HC1 and HC2 constructs include sufficient cis-actingsequences for somatic mutation to occur in the transgenic-JHD mice, weisolated and partially sequenced cDNA clones derived from twoindependent HC1 transgenic lines and one HC2 line. We find that some ofthe γ1 transcripts from transgenic-JHD mice contain V regions withextensive somatic mutations. The frequency of these mutated transcriptsappears to increase with repeated immunizations. FIGS. 66A and 66B showtwo sets of cDNA sequences: one set is derived form an HC1 (line 26)transgenic-JHD mouse that we immunized with a single injection ofantigen 5 days before we isolated RNA; the second set is derived from anHC1 (line 26) transgenic-JHD mouse that we hyperimmunized by injectingantigen on three different days beginning 5 months before we isolatedRNA; the second set is derived from an HC1 (line 26) transgenic-JHDmouse that we hyperimmunized by injecting antigen on three differentdays beginning 5 months before we isolated RNA. Only 2 of the 13 Vregions from the 5 day post-exposure mouse contain any non-germlineencoded nucleotides. Each of these V's contains only a single nucleotidechange, giving an overall somatic mutation frequency of less than 0.1%for this sample. In contrast, none of the 13 V sequences from thehyperimmunized animal are completely germline, and the overall somaticmutation frequency is 1.6%.

Comparison of μ and γ1 transcripts isolated from a single tissue sampleshows that the frequency of somatic mutations is higher in transgenecopies that have undergone a class switch. We isolated and partiallysequenced 47 independent μ and γ1 cDNA clones from a hyperimmunized CH1line 57 transgenic-JHD mouse (FIGS. 67A and 67B). Most of the μ cDNAclones are unmodified relative to the germline sequence, while over halfof the γ1 clones contain multiple non-germline encoded nucleotides. Theγ1 expressing cells are distinct from the μ expressing cells and, whilethe two processes are not necessarily linked, class switching andsomatic mutation are taking place in the same sub-population of B cells.

Although we do not find extensive somatic mutation of the VH251 gene innon-hyperimmunized CH1 transgenic mice, we have found considerablesomatic mutation in VH56p1 and VH51p1 genes in a naive HC2 transgenicmouse. We isolated spleen and lymph node RNA from an unimmunized 9 weekold female HC2 transgenic animal. We individually amplified γ1transcripts that incorporate each of the four V regions in the HC2transgene using V and γ1 specific primers. The relative yields of eachof the specific PCR products were VH56p1>>VH51p1>VH4.21>VH251. Althoughthis technique is not strictly quantitative, it may indicate a bias in Vsegment usage in the HC2 mouse. FIG. 68 shows 23 randomly picked γ1 cDNAsequences derived from PCR amplifications using an equimolar mix of allfour V specific primers. Again we observe a bias toward VH56p1 ( 19/23clones). In addition, the VH56p1 sequences show considerable somaticmutation, with an overall frequency of 2.1% within the V gene segment.Inspection of the CDR3 sequences reveals that although 17 of the 19individual VH56p1 clones are unique, they are derived from only 7different VDJ recombination events. It thus appears that the VH56p1expressing B cells are selected, perhaps by an endogenous pathogen orself antigen, in the naive animal. It may be relevant that this samegene is over-represented in the human fetal repertoire.

Summary

Upstream cis-acting sequences define the functionality of the individualswitch regions, and are necessary for class switching. Ourobservation—that class switching within the HC1 transgene is largelyconfined to cells involved in secondary response, and does not occurrandomly across the entire B cell population—suggests that the minimalsequences contained with the transgene are sufficient. Because the γsequences included in this construct begin only 116 nucleotides upstreamof the start site of the γ1 sterile transcript, the switch regulatoryregion is compact.

Our results demonstrate that these important cis-acting regulatoryelements are either closely linked to individual γ genes, or associatedwith the 3′ heavy chain enhancer included in the HC1 and HC2 transgenes.Because the HC1 and HC2 inserts undergo transgene-autonomous classswitching—which can serve as a marker for sequences that are likely tohave been somatically mutated—we were able to easily find hypermutatedtranscripts that did not originate from translocations to the endogenouslocus. We found somatically mutated γ transcripts in three independenttransgenic lines (two HC1 lines and one HC2 line). It is thereforeunlikely that sequences flanking the integration sites of the transgeneaffect this process; instead, the transgene sequences are sufficient todirect somatic mutation.

Example 36

This example describes the generation of hybridomas from mice homozygousfor an inactivated endogenous immunoglobulin locus and containingtransgene sequences encoding a human sequence heavy chain and humansequence light chain. The hybridomas described secrete monoclonalantibodies comprising a human sequence heavy chain and a human sequencelight chain and bind to a predetermined antigen expressed on Tlymphocytes. The example also demonstrates the capacity of the mice tomake a human sequence antibody in response to a human-derived immunogen,human CD4, and the suitability of such mice as a source for makinghybridomas secreting human sequence monoclonal antibodies reactive withhuman antigens.

A. Generation of Human Ig Monoclonal Antibodies Derived from HC1Transgenic Mice Immunized with a Human CD4 Antigen

A transgenic mouse homozygous for a functionally disrupted J_(H) locusand harboring a transgene capable of rearranging to encode a humansequence heavy chain and a transgene capable of rearranging to encode ahuman sequence light chain was immunized. The genotype of the mouse wasHC1-26⁺ KC1e-1536⁺ J_(H)D⁺/J_(H)D⁻, indicating homozygosity for murineheavy chain inactivation and the presence of germline copies of the HC1human sequence heavy chain transgene and the KC1e human sequence lightchain transgene.

The mouse was immunized with a variant of the EL4 cell line (ATCC)expressing a mouse-human hybrid CD4 molecule encoded by a stablytransfected polynucleotide. The expressed CD4 molecule comprises asubstantially human-like CD4 sequence. Approximately 5×10⁶ cells in 100μl of PBS accompanied by 100 μl of Complete Freund's Adjuvant (CFA) wereintroduced into the mouse via intraperitoneal injection on Day 0. Theinoculation was repeated on Days 7, 14, 21, 28, 60, and 77, with testbleeds on Days 18, 35, and 67. The spleen was removed on Day 81 andapproximately 7.2×10⁷ spleen cells were fused to approximately 1.2×10⁷fusion partner cells (P3x63Ag8.653 cell line; ATCC) by standard methods(PEG fusion) and cultured in RPMI 1640 15% FCS, 4 mM glutamine, 1 mMsodium pyruvate plus HAT and PSN medium. Multiple fusions wereperformed.

Hybridomas were grown up and supernatants were tested with ELISA forbinding to a commercial source of purified recombinant soluble humansequence CD4 expressed in CHO cells (American Bio-Technologies, Inc.(ABT), Cambridge, Mass.) and/or CD4 obtained from NEN-DuPont. The ABTsample contained a purified 55 kD human CD4 molecule comprised the V₁through V₃ domains of human CD4. The recombinant human sequence CD4(produced in CHO-K1 cells) was adsorbed to the assay plate and used tocapture antibody from hybridoma supernatants, the captured antibodieswere then evaluated for binding to a panel of antibodies which bindeither human μ, human κ, human γ, murine μ, or murine κ.

One hybridoma was subcloned from its culture plate well, designated 1F2.The 1F2 antibody bound to the ABT CD4 preparation, was positive forhuman μ and human κ, and was negative for human γ, mouse γ, and mouse κ.

B. Generation of Human Ig Monoclonal Antibodies Derived from HC2Transgenic Mice Immunized with Human CD4 and Human IgE.

The heavy chain transgene, HC2, is shown in FIG. 56 and has beendescribed supra (see, Example 34).

The human light chain transgene, KCo4, depicted in FIG. 56 is generatedby the cointegration of two individually cloned DNA fragments at asingle site in the mouse genome. The fragments comprise 4 functional Vκsegments, 5J segments, the Cκ exon, and both the intronic and downstreamenhancer elements (see Example 21) (Meyer and Neuberger (1989), EMBO J.8:1959-1964; Judde and Max (1992), Mol. Cell Biol. 12:5206-5216).Because the two fragments share a common 3 kb sequence (see FIG. 56),they can potentially integrate into genomic DNA as a contiguous 43 kbtransgene, following homologous recombination between the overlappingsequences. It has been demonstrated that such recombination eventsfrequently occur upon microinjection of overlapping DNA fragments(Pieper et al. (1992), Nucleic Acids Res. 20:1259-1264). Co-injectedDNA's also tend to co-integrate in the zygote, and the sequencescontained within the individually cloned fragments would subsequently bejointed by DNA rearrangement during B cell development. Table 12 showsthat transgene inserts from at least 2 of the transgenic lines arefunctional. Examples of VJ junctions incorporating each of the 4transgene encoded V segments, and each of the 5J segments, arerepresented in this set of 36 clones.

TABLE 11 Human variable region usage in hybridomas Subclone SpecificityIsotype Vh Dh Jh Vκ Jκ 2C11.8 nCD4 IgMκ 251 nd.* nd. nd. nd. 2C5.1 rCD4IgGκ 251 HQ52 JHS 65.15 JK4 4E4.2 rCD4 IgGκ 251 HQ52 JHS 65.15 JK4*n.d., not determined

Human light chain V and J segment usage in KCo4 transgenic mice. Thetable shows the number of PCR clones, amplified from cDNA derived fromtwo transgenic lines, which contain the indicated human kappa sequences.cDNA was synthesized using spleen. RNA isolated from w individual KCo4transgenic mice (mouse #8490, 3 mo., male, KCo4 line 4437; mouse #8867,2.5 mo., female, KCo4 line 4436). The cDNA was amplified by PCR using aCκ specific oligonucleotide. 5′TAG AAG GAA TTC AGC AGG CAC ACA ACA GAGGCA GTT CCA3′ (SEQ ID NO.173), 1:3 mixture of the following 2 Vκoligonucleotides: 5′ AGC TTC TCG AGC TCC TGC AND TGC A TCT GTT TCC CAGGTG CC 3′ (SEQ ID NO.174) and 5′ CAG CTT CTC GAG CTC CTG CTA CTC TGGCTC(C,A)CA GAT ACC 3′ (SEQ ID NO.175). The PCR product was digested withXhoI and EcoRI, and cloned into a plasmid vector. Partial nucleotidesequences were determined by the dideoxy chain termination method for 18randomly picked clones from each animal. The sequences of each clonewere compared to the germline sequence of the unrearranged transgene.

Twenty-three light chain minilocus positive and 18 heavy chain positivemice developed from the injected embryos. These mice, and their progeny,were bred with mice containing targeted mutations in the endogenousmouse heavy (strain JHD) and κ light chain loci (strain JCKD) to obtainmice containing human heavy and κ light chain in the absence offunctional mouse heavy and κ light chain loci. In these mice, the onlymouse light chain contribution, if any, is from the mouse λ locus.

Table 13 show that somatic mutation occurs in the variable regions ofthe transgene-encoded human heavy chain transcripts of the transgenicmice. Twenty-three cDNA clones from a HC2 transgenic mouse werepartially sequenced to determine the frequency of non-germline encodednucleotides within the variable region. The data include only thesequence of V segment codons 17-94 from each clone, and does not includeN regions. RNA was isolated from the spleen and lymph node of mouse 5250(HC2 line 2550 hemizygous, JHD homozygous). Single-stranded cDNA wassynthesized and γ transcripts amplified by PCR as described[references]. The amplified cDNA was cloned into plasmid vectors, and 23randomly picked clones were partially sequenced by the dideoxychain-termination method. The frequency of PCR-introduced nucleotidechanges is estimated from constant region sequence as <0.2%.

TABLE 13 The Variable Regions of Human γ Transcripts in HC2 TransgenicMice Contain Non-Germline-Encoded Nucleotides Number of non- Frequencyof non- VH Number of germline encoded germline-encoded Segment clonesnucleotides nucleotides (%) VH251 0 — VH56P1 10 100 2.1 VH51P1 1 5 2.0VH4.21 3 0 0.0Flow Cytometry

We analyzed the stained cells using a FACScan flow cytometer and LYSISII software (Becton Dickinson, San Jose, Calif.). Spleen cells werestained with the following reagents: propidium iodide (Molecular Probes,Eugene, Oreg.), phycoerythrin conjugated α-human Igκ (clone HP6062;Caltag, S. San Francisco, Calif.), phycoerythrin conjugated α-mouse Igκ(clone X36; Becton Dickinson, San Jose, Calif.), FITC conjugated a-mouseIgλ (clone R26-46; Pharmingen, San diego, Calif.), FITC conjugatedα-mouse Igμ (clone R6-60.2; Pharmingen, San Diego, Calif.), FITCconjugated α-human Igμ (clone G20-127; Pharmingen, San Diego, Calif.),and Cy-Chrome conjugated α-mouse B220 (clone RA3-6B2; Pharmingen, SanDiego, Calif.).

Expression of Human Ig Transgenes

FIG. 69 shows a flow cytometric analysis of spleen cells from KCo4 andHC2 mice that are homozygous for both the JHD and JCKD mutations. Thehuman sequence HC2 transgene rescued B cell development in the JHDmutant background, restoring the relative number of B220⁺ cells in thespleen to approximately half that of a wild type animal. These B cellsexpressed cell surface immunoglobulin receptors that used transgeneencoded heavy chain. The human KCo4 transgene was also functional, andcompeted successfully with the intact endogenous λ light chain locus.Nearly 95% of the splenic B cells in JHD/JCKD homozygous mutant micethat contain both heavy and light chain human transgenes (doubletransgenic) expressed completely human cell surface IgMκ.

Serum Ig levels were determined by ELISA done as follows: human μ:microtiter wells coated with mouse Mab a human IgM (clone CH6, TheBinding Site, Birmingham, UK) and developed with peroxidase conjugatedrabbit α human IgM(fc) (cat #309-035-095, Jackson Immuno Research, WestGrove, Pa.). Human γ: microtiter wells coated with mouse MAb a humanIgG1 (clone HP6069, Calbiochem, La Jolla, Calif.) and developed withperoxidase conjugated goat α human IgG(fc) (cat #109-036-098, JacksonImmuno Research, West Grove, Pa.). Human κ: microtiter wells coated withmouse Mab α human IgK (cat #0173, AMAC, Inc. Igκ (cat #A7164, SigmaChem. Co., St. Louis, Mo.). Mouse γ: microtiter wells coated with goat amouse IgG (cat 1115-006-071, Jackson Immuno Research, West Grove, Pa.).Mouse λ: microtiter wells coated with rat MAb α mouse Igλ (cat #02171D,Pharmingen, San Diego, Calif.) and developed with peroxidase conjugatedrabbit a mouse IgM(fc) (cat #309-035-095, Jackson Immuno Research, WestGrove, Pa.). Bound peroxidase is detected by incubation with hydrogenperoxide and 2,2′-Azino-bis-)₃-Ethylbenzthiazoline-6-Sulfonic Acid,Sigma Chem. Co., St. Louis, Mo.). The reaction product is measured byabsorption at 415 nm.

The double transgenic mice also express fully human antibodies in theserum. FIG. 70 shows measured serum levels of immunoglobulin proteinsfor 18 individual double transgenic mice, homozygous for endogenousheavy and kappa light chain inactivations, derived from severaldifferent transgenic founder animals. We found detectable levels ofhuman μ, γ1, and κ. We have shown supra that the expressed human γ1results from authentic class switching by genomic recombination betweenthe transgene μ and γ1 switch regions. Furthermore, we have found thatintra-transgene class switching was accompanied by somatic mutation ofthe heavy chain variable regions. In addition to human immunoglobulins,we also found mouse γ and λ in the serum. The present of mouse λ proteinis expected because the endogenous locus is completely intact. We haveshown elsewhere that the mouse γ expression is a consequence oftrans-switch recombination of transgene VDJ segments into the endogenousheavy chain locus. This trans-switching phenomenon, which was originallydemonstrated for wild-type heavy chain alleles and rearranged VDJtransgenes (Durdik et al. (1989), Proc. Natl. Acad. Sci. USA86:2346-2350; Gerstein et al. (1990), Cell 63:537-548), occurs in themutant JHD background because the downstream heavy chain constantregions and their respective switch elements are still intact.

The serum concentration of human IgMκ in the double transgenic mice wasapproximately 0.1 mg/ml, with very little deviation between animals orbetween lines. However, human γ1, mouse γ, and mouse λ levels range from0.1 to 10 micrograms/ml. The observed variation in γ levels betweenindividual animals may be a consequence of the fact that γ is aninducible constant region. Expression presumably depends on factors suchas the health of the animal, exposure to antigens, and possibly MHCtype. The mouse λ serum levels are the only parameter that appears tocorrelate with individual transgenic lines. KCo4 line 4436 mice whichhave the fewest number of copies of the transgene per integration(approximately 1-2 copies) have the highest endogenous λ levels, whileKCo4 line 4437 mice (^(˜)10 copies per integration) have the lowest λlevels. This is consistent with a model in which endogenous λ rearrangessubsequent to the κ transgene, and in which the serum λ level is notselected for, but is instead a reflection of the relative size of theprecursor B cell pool. Transgene loci containing multiple light chaininserts may have the opportunity to undergo more than one V to Jrecombination event, with an increased probability that one of them willbe functional. Thus high copy lines will have a smaller pool ofpotential λ cells.

Immunizations with Human CD4 and IgE

To test the ability of the transgenic B cells to participate in animmune response, we immunized double transgenic mice with human proteinantigens, and measured serum levels of antigen specific immunoglobulinsby ELISA. Mice were immunized with 50 μg recombinant sCD4 (cat. #013101,American Bio-Technologies Inc., Cambridge, Mass.) covalently linked topolystyrene beads (cat #08226, Polysciences Inc., Warrington, Pa.) incomplete Freund's adjuvant by intraperitoneal injection. Each of themice are homozygous for disruptions of the endogenous μ and κ loci, andhemizygous for the human heavy chain transgene HC2 line 2500 and human κlight chain transgene KCo4 line 4437.

Methods

Serum samples were diluted into microtiter wells coated with recombinantsCD4. Human antibodies were detected with peroxidase conjugated rabbit ahuman IgM(fc) (Jackson Immuno Research, West Grove, Pa.) or peroxidaseconjugated goat anti-human Igκ (Sigma, St. Louis, Mo.).

FIG. 71A shows the primary response of transgenic mice immunized withrecombinant human soluble CD4. All four of the immunized animals show anantigen-specific human IgM response at one week. The CD4-specific serumantibodies comprise both human μ heavy chain and human κ light chain.

To evaluate the ability of the HC2 transgene to participate in asecondary response, we hyperimmunized the transgenic mice by repeatedinjection with antigen, and monitored the heavy chain isotype of theinduced antibodies. Mice homozygous for the human heavy chain transgeneHC2 and human κ light chain transgene KCo4 were immunized with 25 μg ofhuman IgEκ (The Binding Site, Birmingham, UK) in complete Freund'sadjuvant on day=0. Thereafter, animals were injected with IgEκ inincomplete Freund's adjuvant at approximately weekly intervals. Serumsamples were diluted 1:10, and antigen-specific ELISAs were performed onhuman IgE, λ coated plates.

FIG. 71B shows a typical time course of the immune response from theseanimals: we injected double transgenic mice with human IgE in completeFreund's adjuvant, followed by weekly boosts of IgE in incompleteFreund's adjuvant. The initial human antibody response was IgMκ,followed by the appearance of antigen specific human IgGκ. The inducedserum antibodies in these mice showed no cross-reactivity to human IgMor BSA. The development, over time, of a human IgG

We have also tested the ability of the heavy chain transgene to undergoclass switching in vitro: splenic B cells purified form animalshemizygous for the same heavy chain construct (HC2, line 2550) switchfrom human IgM to human IgG1 in the presence of LPS and recombinantmouse IL-4. However, in vitro switching did not take place in thepresence of LPS and recombinant mouse IL-2, or LPS alone.

We find human IgM-expressing cells in the spleen, lymph nodes,peritoneum, and bone marrow of the double-transgenic/double-knockout(0011) mice. Although the peritoneal cavity contains the normal numberof B cells, the absolute number of transgenic B cells in the bone marrowand spleen is approximately 10-50% of normal. The reduction may resultfrom a retardation in transgene-dependent B cell development. Thedouble-transgenic/double-knockout (0011) mice also express fully humanantibodies in the serum, with significant levels of human μ, γ1, and κin these mice. The expressed human γ1 results from authentic classswitching by genomic recombination between the transgene μ and γ1 switchregions. Furthermore, the intratransgene class switching is accompaniedby somatic mutation of the heavy chain variable regions encoded by thetransgene. In addition to human immunoglobulins, we find mouse μ andmouse λ in these mice. The mouse μ expression appears to be a result oftrans-switching recombination, wherein transgene VDJ gene is recombinedinto the endogenous mouse heavy chain locus. Trans-switching, which wasoriginally observed in the literature for wild-type heavy chain allelesand rearranged VDJ transgenes, occurs in our J_(H) ⁻/⁻ backgroundbecause the mouse downstream heavy chain constant regions and theirrespective switch elements are still intact.

To demonstrate the ability of the transgenic B cells to participate inan immune response, we immunized the 0011 mice with human proteinantigens, and monitored serum levels of antigen-specificimmunoglobulins. The initial human antibody response is IgM, followed bythe expression of antigen-specific human IgG (FIG. 71B and FIG. 73). Thelag before appearance of human IgG antibodies is consistent with anassociation between class-switching and a secondary response to antigen.

In a transgenic mouse immunized with human CD4, human IgG reactivity tothe CD4 antigen was detectable at serum concentrations ranging from2×10⁻² to 1.6×10⁻⁴.

Identification of Anti-Human CD4 Hybridomas

A transgenic mouse homozygous for the human heavy chain transgene HC2and human κ light chain transgene KCo4 were immunized with 20 μg ofrecombinant human CD4 in complete Freund's adjuvant on day 0.Thereafter, animals were injected with CD4 in incomplete Freund'sadjuvant at approximately weekly intervals. FIG. 73 shows human antibodyresponse to human CD4 in serum of the transgenic mouse. Serum sampleswere diluted 1:50, and antigen-specific ELISAs were performed on humanCD4 coated plates. Each line represents individual sampledeterminations. Solid circles represent IgM, open squares represent IgG.

We also isolated hybridoma cell lines from one of the mice thatresponded to human CD4 immunization. Five of the cloned hybridomassecrete human IgGκ (human γ1/human κ) antibodies that bind torecombinant human CD4 and do not crossreact (as measured by ELISA) witha panel of other glycoprotein antigens. The association and dissociationrates of the immunizing human CD4 antigen for the monoclonal antibodiessecreted by two of the IgGK hybridomas, 4E4.2 and 2C5.1, weredetermined. The experimentally-derived binding constants (K_(a)) wereapproximately 9×10⁻⁷ M⁻¹ and 8×10⁻⁷ M⁻¹ for antibodies 4E4.2 and 2C5.1,respectively. These K_(a) values fall within the range of murine IgGanti-human CD4 antibodies that have been used in clinical trials byothers (Chen et al. (1993) Int. Immunol. 6: 647).

A mouse of line #7494 (0012;HC1-26+;JHD++;JKD++;KC2-1610++) wasimmunized on days 0, 13, 20, 28, 33, and 47 with human CD4, and producedanti-human CD4 antibodies comprised of human κ and human μ or γ.

By day 28, human μ and human κ were found present in the serum. By day47, the serum response against human CD4 comprised both human μ andhuman γ, as well as human κ. On day 50, splenocytes were fused withP3X63-Ag8.653 mouse myeloma cells and cultured. Forty-four out of 700wells (6.3%) contained human γ and/or κ anti-human CD4 monoclonalantibodies. Three of these wells were confirmed to contain human γanti-CD4 monoclonal antibodies, but lacked human κ chains (presumablyexpressing mouse λ). Nine of the primary wells contained fully humanIgMκ anti-CD4 monoclonal antibodies, and were selected for furthercharacterization. One such hybridoma expressing fully human IgMκanti-CD4 monoclonal antibodies was designated 2C11-8.

Primary hybridomas were cloned by limiting dilution and assessed forsecretion of human μ and κ monoclonal antibodies reactive against CD4.Five of the nine hybridomas remained positive in the CD4 ELISA. Thespecificity of these human IgMκ monoclonal antibodies for human CD4 wasdemonstrated by their lack of reactivity with other antigens includingovalbumin, bovine serum albumin, human serum albumin, keyhole limpethemacyanin, and carcinoembryonic antigen. To determine whether thesemonoclonal antibodies could recognize CD4 on the surface of cells (i.e.,native CD4), supernatants from these five clones were also tested forreactivity with a CD4+ T cell line, Sup T1. Four of the five human IgMκmonoclonal antibodies reacted with these CD4+ cells. To further confirmthe specificity of these IgMκ monoclonal antibodies, freshly isolatedhuman peripheral blood lymphocytes (PBL) were stained with theseantibodies. Supernatants from clones derived from four of the fiveprimary hybrids bound only to CD4+ lymphocytes and not to CD8+lymphocytes (FIG. 72).

FIG. 72 shows reactivity of IgMκ anti-CD4 monoclonal antibody with humanPBL. Human PBL were incubated with supernatant from each clone or withan isotype matched negative control monoclonal antibody, followed byeither a mouse anti-human CD4 monoclonal antibody conjugated to PE (toprow) or a mouse anti-human CD8 Ab conjugated to FITC (bottom row). Anybound human IgMκ was detected with a mouse anti-human μ conjugated toFITC or to PE, respectively. Representative results for one of theclones, 2C11-8 (right side) and for the control IgMκ (left side) areshown. As expected, the negative control IgMκ did not react with T cellsand the goat anti-human μ reacted with approximately 10% of PBL, whichwere presumably human B cells.

Good growth and high levels of IgMκ anti-CD4 monoclonal antibodyproduction are important factors in choosing a clonal hybridoma cellline for development. Data from one of the hybridomas, 2C11-8, showsthat up to 5 pg/cell/d can be produced (FIG. 74). Similar results wereseen with a second clone. As is commonly observed, production increasesdramatically as cells enter stationary phase growth. FIG. 74 shows cellgrowth and human IgMκ anti-CD4 monoclonal antibody secretion in smallscale cultures. Replicate cultures were seeded at 2×10⁵ cells/ml in atotal volume of 2 ml. Every twenty-four hours thereafter for four days,cultures were harvested. Cell growth was determined by counting viablecells and IgMκ production was quantitated by an ELISA for total human μ(top panel). The production per cell per day was calculated by dividingthe amount of IgMκ by the cell number (bottom panel).

FIG. 75 shows epitope mapping of a human IgMκ anti-CD4 monoclonalantibody. Competition binding flow cytometric experiments were used tolocalize the epitope recognized by the IgMκ anti-CD4 monoclonalantibody, 2C11-8. For these studies, the mouse anti-CD4 monoclonalantibodies, Leu3a and RPA-T4, which bind to unique, nonoverlappingepitopes on CD4 were used. PE fluorescence of CD4+ cells preincubatedwith decreasing concentrations of either RPA-TA or Leu-3a followed bystaining with 2C11-8 detected with PE-conjugated goat anti-human IgM.There was concentration-dependent competition for the binding of thehuman IgMκ anti-CD4 monoclonal antibody 2C11-8 by Leu3a but not byRPA-T4 (FIG. 75). Thus, the epitope recognized by 2C11-8 was similar toor identical with that recognized by monoclonal antibody Leu3a, butdistinct from that recognized by RPA-T4.

In summary, we have produced several hybridoma clones that secrete humanIgMκ monoclonal antibodies that specifically react with native human CD4and can be used to discriminate human PBLs into CD4⁺ and CD4⁻subpopulations. At least one of these antibodies binds at or near theepitope defined by monoclonal antibody Leu3a. Monoclonal antibodiesdirected to this epitope have been shown to inhibit a mixed leukocyteresponse (Engleman et al., J. Exp. Med. (1981) 153:193). A chimericversion of monoclonal antibody Leu3a has shown some clinical efficacy inpatients with mycosis fungoides (Knox et al. (1991) Blood 77:20).

We have isolated cDNA clones from 3 different hybridoma cell lines(2C11.8, 2C5.1, and 4E4.2), and have determined the partial nucleotidesequence of some of the expressed immunoglobulin genes in each of thesecell lines. For sequence analysis, total RNA was isolated fromapproximately 5×10⁶ hybridoma cells. sscDNA was synthesized by primingreverse transcription with oligo dT. A portion of this sscDNA was usedin duplicate PCR reactions primed by a pool of oligos with specificitiesfor either (i) heavy chain variable framework regions contained withinthe HC1 or HC2 transgenes and a single downstream oligo specific forconstant human gamma sequence, or (ii) light chain variable frameworkregions contained within the KC2 or KCo4 transgene and a singledownstream oligo specific for constant human kappa sequence. Productsfrom these PCR reactions were digested with appropriate restrictionenzymes, gel purified, and independently cloned into pNNO3 vector. DNAwas isolated and manual dideoxy and/or automated fluorescent sequencingreactions performed on dsDNA.

The characteristics of the three hybridomas, 2C11.8, 2C5.1, and 4E4.2,are given below in Table 11.

TABLE 11 Human variable region usage in hybridomas Subclone SpecificityIsotype Vh Dh Jh Vκ Jκ 2C11.8 nCD4 IgMκ 251 nd.* nd. nd. nd. 2C5.1 rCD4IgGκ 251 HQ52 JHS 65.15 JK4 4E4.2 rCD4 IgGκ 251 HQ52 JHS 65.15 JK4*n.d., not determined

Nucleotide sequence analysis of expressed heavy and light chainsequences from the two IgGκ hybridomas 2C5.1 and 4E4.2 reveal that theyare sibling clones derived from the same progenitor B cell. The heavyand light chain V(D)J junctions from the two clones are identical,although the precise nucleotide sequences differ by presumptive somaticmutations. The heavy chain VDJ junction sequence is:

     VH251             N         DHQ52        JH5 SEQ. ID No.: 176 TATTAC TGT GCG AG (g gct cc) A ACT GGG C TGG TTC                              GA        GAC Y   Y   C   A   RA    P         T   G     W   F                               D         D SEQ. ID No.: 177

The light chain VJ junction is:

       Vk65.15          N     Jk4 (SEQ ID NO. 178) TAT AAT AGT TAC CCTCC (t) ACT TTC GGC  Y   N   S   Y   P   P      T   F   G (SEQ ID NO.179)

The following non-germline encoded codons were identified (presumptivesomatic mutations):

2CS.1 heavy AGC -> AGG S28R (replacement) chain light CCG -> ACG P119T(replacement) chain 4E4.2 heavy AGC -> AGG S28R (replacement) chain CTG-> CTA L80L (silent) light GAG -> GAC E41D (replacement) chain AGG-> AAG R61K (replacement) CCG -> ACG P119T (replacement)

We conclude that these two gamma hybridomas are derived from B cellsthat have undergone a limited amount of somatic mutation. This datashows that the HC2 transgenic animals use the VH5-51 (aka VH251) Vsegment. We have previously shown that VH4-34, VH1-69, and VH3-30.3 areexpressed by these mice. The combination of these results demonstratesthat the HC2 transgenic @ce express all four of the transgene encodedhuman VH genes.

We conclude that human immunoglobulin-expressing B cells undergodevelopment and respond to antigen in the context of a mouse immunesystem. Antigen responsivity leads to immunoglobulin heavy chain isotypeswitching and variable region somatic mutation. We have alsodemonstrated that conventional hybridoma technology can be used toobtain monoclonal human sequence antibodies from these mice. Therefore,these transgenic mice represent a source of human antibodies againsthuman target antigens.

Example 37

This example describes the generation of transgenic mice homozygous foran inactivated endogenous heavy chain and κ chain locus and harboring atransgene capable of isotype switching to multiple downstream humanC_(H) genes. The example also demonstrates a cloning strategy forassembling large transgenes (e.g., 160 kb) by co-microinjection ofmultiple DNA fragments comprising overlapping homologous sequence joints(see FIG. 76), permitting construction of a large transgene from morethan two overlapping fragments by homologous recombination of aplurality of homology regions at distal ends of the set of fragments tobe assembled in vivo, such as in a microinjected ES cell or its clonalprogeny. The example also shows, among other things, that isolatedlymphocytes from the transgenic animals can be induced to undergoisotype switching in vitro, such as with IL-4 and LPS.

A set of five different plasmid clones was constructed such that theplasmid inserts could be isolated, substantially free of vectorsequences; and such that the inserts together form a single imbricateset of overlapping sequence spanning approximately 150 kb in length.This set includes human V, D, J, μ, γ3, and γ1 coding sequences, as wellas a mouse heavy chain 3′ enhancer sequence. The five clones are, in 5′to 3′ order: pH3V4D, pCOR1xa, p11-14, pP1-570, and pHP-3a (FIG. 76).Several different cloning vectors were used to generate this set ofclones. Some of the vectors were designed specifically for the purposeof building large transgenes. These vectors (pGP1a, pGP1b, pGP1c, pGP1d,pGP1f, pGP2a, and pGP2b) are pBR322-based plasmids that are maintainedat a lower copy number per cell than the pUC vectors (Yanisch-Perron etal. (1985) Gene 33: 103-119). The vectors also include trpAtranscription termination signals between the polylinker and the 3′ endof the plasmid .beta.-lactamase gene. The polylinkers are flanked byrestriction sites for the rare-cutting enzyme NotI; thus allowing forthe isolation of the insert away from vector sequences prior to embryomicroinjection. Inside of the NotI sites, the polylinkers include uniqueXhoI and SalI sites at either end. The pGP1 vectors are described inTaylor et al. (1992) Nucleic Acids Res. 23: 6287. To generate the pGP2vectors, pGP1f was first digested with AlwNI and ligated with thesynthetic oligonucleotides o-236 and o-237 (o-236, 5′-ggc gcg cct tggcct aag agg cca-3′(SEQ ID NO. 180); o-237, 5′-cct ctt agg cca agg cgcgcc tgg-3′) (SEQ ID NO. 181) The resulting plasmid is called pGP2a.Plasmid pGP2a was then digested with KpnI and EcoRI, and ligated withthe oligonucleotides o-288 and o-289 (o-288, 5′-aat tca gta tcg atg tggtac-3′(SEQ ID NO. 182); o-289, 5′-cac atc gat act g-3′(SEQ ID NO. 183)to create pGP2b (FIG. 77A and FIG. 77B).

The general scheme for transgene construction with the pGP plasmids isoutlined in FIG. 78 (paths A and B). All of the component DNA fragmentsare first cloned individually in the same 5′ to 3′ orientation in pGPvectors. Insert NotI, XhoI and SalI sites are destroyed byoligonucleotide mutagenesis or if possible by partial digestion,polymerase fill-in, and blunt end ligation. This leaves only thepolylinker derived XhoI and SalI sites at the 5′ and 3′ ends of eachinsert. Individual inserts can then be combined stepwise by the processof isolating XhoI/SalI fragments from one clone and inserting theisolated fragment into either the 5′ XhoI or 3′ SalI site of anotherclone (FIG. 78, path A). Transformants are then screened by filterhybridization with one or more insert fragments to obtain the assembledclone. Because XhoI/SalI joints cannot be cleaved with either enzyme,the resulting product maintains unique 5′ XhoI and 3′ SalI sites, andcan be used in the step of the construction. A variation of this schemeis carried out using the vectors pGP2a and pGP2b (FIG. 78, path B).These plasmids includes an SfiI site between the ampicillin resistancegene and the plasmid origin of replication. By cutting with SfiI andXhoI or SalI, inserts can be isolated together with either the drugresistance sequence or the origin of replication. One SfiI/XhoI fragmentis ligated to one SfiI/SalI fragment in each step of the synthesis.There are three advantages to this scheme: (i) background transformantsare reduced because sequences from both fragments are required forplasmid replication in the presence of ampicillin; (ii) the ligation canonly occur in a single 5′ to 3′ orientation; and (iii) the SfiI ends arenot self-compatible, and are not compatible with SalI or XhoI, thusreducing the level of non-productive ligation. The disadvantage of thisscheme is that insert SfiI sites must be removed as well as NotI, XhoI,and SalI sites. These medium copy vectors are an improvement over thecommonly used pUC derived cloning vectors. To compare the ability ofthese vectors to maintain large DNA inserts, a 43 kb XhoI fragmentcomprising the human JH/Cμ region was ligated into the SalI site ofpSP72 (Promega, Madison, Wis.), pUC19 (BRL, Grand Island, N.Y.), andpGP1f. Transformant colonies were transferred to nitrocellulose andinsert containing clones were selected by hybridization withradiolabeled probe. Positive clones were grown overnight in 3 ml mediaand DNA isolated: EcoRI digestion of the resulting DNA reveals that allthe pSP72 and pUC19 derived clones deleted the insert (FIG. 79);however, 12 of the 18 pGP1f derived clones contained intact inserts.Both orientations are represented in these 12 clones.

The construction and isolation of the five clones (pH3V4D, pCOR1xa,p11-14, pP1-570, and pHP-3a) used to generate the HCo7 transgene isoutlined below.

pH3V4D.

Germline configuration heavy chain variable gene segments were isolatedfrom phage 1 genomic DNA libraries using synthetic oligonucleotideprobes for VH1 and VH3 classes. The VH1 class probe was o-49:

(SEQ ID No. 78) 5′-gtt aaa gag gat ttt att cac ccc tgt gtc ctc tcc acaggt gtc-3′

The VH3 class probe was o-184:

(SEQ ID NO. 184) 5′-gtt tgc agg tgt cca gtg t(c,g)a ggt gca gct g(g,t)tgga gtc(t,c)(g,c)g-3′

Positively hybridizing clones were isolated, partially restrictionmapped, subcloned and partially sequenced. From the nucleotide sequenceit was determined that one of the VH1 clones isolated with the o-49probe encoded a VH gene segment, 49.8, comprising an amino acid sequenceidentical to that contained in the published sequence of the hv1263 gene(Chen et al. (1989) Arthritis Rheum. 32: 72). Three of the VH3 genes,184.3, 184.14, and 184.17, that were isolated with the o-184 probecontained sequences encoding identical amino acid sequences to thosecontained in the published for the VH genes DP-50, DP-54, and DP-45(Tomlinson et al. (1992) J. Mol. Biol. 227: 776). These four VH geneswere used to build the pH3V4D plasmid.

The 184.3 gene was found to be contained within a 3 kb BamHI fragment.This fragment was subcloned into the plasmid vector pGP1f such that theXhoI site of the polylinker is 5′ of the gene, and the SalI site is 3′.The resulting plasmid is called p184.3.36f. The 184.14 gene was found tobe contained within a 4.8 kb HindIII fragment. This fragment wassubcloned into the plasmid vector pUC19 in an orientation such that thegene could be further isolated as a 3.5 kb fragment by XhoI/SalIdigestion at a genomic XhoI site 0.7 kb upstream of the gene and apolylinker derived SalI site 3′ of the gene. The resulting plasmid iscalled p184.14.1. The 184.17 gene was found to be contained within a 5.7kb HindIII fragment. This fragment was subcloned into the plasmid vectorpSP72 (Promega, Madison, Wis.) in an orientation such that thepolylinker derived XhoI and SalI sites are, respectively, 5′ and 3′ ofthe gene. The insert of this plasmid includes an XhoI site at the 3′ endof the gene which was eliminated by partial digestion with XhoI, Klenowfragment filling-in, and religation. The resulting plasmid is calledp184.17SK. The 49.8 gene was found to be contained within 6.3 kb XbaIfragment. This fragment was subcloned into the plasmid vector pNNO3,such that the polylinker derived XhoI and ClaI sites are, respectively,5′ and 3′ of the gene, to create the plasmid pVH49.8 (Taylor et al.(1994) International Immunol. 6: 579). The XhoI/ClaI insert of pVH49.8was then subcloned into pGP1f to create the plasmid p49.8f, whichincludes unique XhoI and SalI sites respectively at the 5′ and 3′ end ofthe 49.8 gene.

The 3.5 kb XhoI/SalI fragment of p184.14.1 was cloned into the XhoI siteof p184.3.36f to generate the plasmid pRMVH1, which includes both the184.14 and the 184.3 genes in the same orientation. This plasmid wasdigested with XhoI and the 5.7 kb XhoI/SalI fragment of p184.17SK wasinserted to create the plasmid pRMVH2, which contains, from 5′ to 3′,the three VH genes 184.17, 184.14, and 184.3, all in the sameorientation. The plasmid pRMVH2 was then cut with XhoI, and the 6.3 kbXhoI/SalI insert of p49.8f inserted to create the plasmid pH3VH4, whichcontains, from 5′ to 3′, the four VH genes 49.8, 184.17, 184.14, and184.3, all in the same orientation.

The 10.6 kb XhoI/EcoRV insert of the human D region clone pDH1(described supra; e.g., in Example 12) was cloned into XhoI/EcoRVdigested pGPe plasmid vector to create the new plasmid pDH1e. Thisplasmid was then digested with EcoRV and ligated with a synthetic linkerfragment containing a SalI site (5′-ccg gtc gac ccg-3′). The resultingplasmid, pDH1es, includes most of the human D1 cluster within an insertthat can be excised with XhoI and SalI, such that the XhoI site is onthe 5′ end, and the SalI site is on the 3′ end. This insert was isolatedand cloned into the SalI site of pH3VH4 to create the plasmid pH3VH4D,which includes four germline configuration human VH gene segments and 8germline configuration human D segments, all in the same 5′ to 3′orientation. The insert of this clone can be isolated, substantiallyfree of vector sequences, by digestion with NotI.

pCOR1xa

The plasmid pCOR1 (described supra) which contains a 32 kb XhoI insertthat includes 9 human D segments, 6 human J segments, the Jμ intronicheavy chain enhancer, the μ switch region, and the Cμ coding exons—waspartially digested with XhoI, Klenow treated, and a synthetic SalIlinker ligated in to produce the new plasmid pCOR1xa, which has a uniqueXhoI site at the 5′ end and a unique SalI site at the 3′ end. Both pCOR1and pCOR1xa contain a 0.6 kb rat heavy chain 3′ enhancer fragment at the3′ end, which is included in the insert if the plasmid is digested withNotI instead of XhoI or XhoI/SalI.

PP1-570

A phage P1 library (Genome Systems Inc., St. Louis, Mo.) was screened byPCR using the oligonucleotide primer pair:

(SEQ ID NO. 186) 5′-tca caa gcc cag caa cac caa g-3′ (SEQ ID NO. 187)5′-aaa agc cag aag acc ctc tcc ctg-3′This primer pair was designed to generate a 216 bp PCR product with ahuman γ gene template. One of the P1 clones identified was found tocontain both the human γ3 and γ1 genes within an 80 kb insert. Theinsert of this clone, which is depicted in FIG. 80, can be isolated,substantially free of vector sequences, by digestion with NotI and SalI.p11-14

Restriction mapping of the human γ3/γ1 clone P1-570 revealed a 14 kbBamHI fragment near the 5′ end of the insert. This 14 kb fragment wassubcloned into the plasmid vector pGP1f such that the polylinker derivedSalI site is adjacent to the 5′ end of the insert. The resulting plasmidis called pB14. Separately, an 11 kb NdeI/SpeI genomic DNA fragmentcovering the 3′ end of the human μ gene and the 5′ end of the human 6gene, derived from the plasmid clone pJ1NA (Choi et al. (1993) NatureGenetics 4: 117), was subcloned into the SalI site of pBluescript(Stratagene, LaJolla, Calif.) using synthetic oligonucleotide adapters.The resulting SalI insert was then isolated and cloned into the SalIsite of pB14 such that the relative 5′ to 3′ orientation of the μfragment from pJ1NA is the same as that of the γ fragment from P1-570.The resulting clone is called p11-14. The insert of this clone can beisolated, substantially free of vector sequences, by digestion withNotI.

PHP-3a

The mouse heavy chain 3′ enhancer (Dariavach et al. (1991) Eur. J.Immunol. 21: 1499; Lieberson et al. (1991) Nucleic Acids Res. 19: 933)was cloned from a balb/c mouse genomic DNA phage λ library. To obtain aprobe, total balb/c mouse thymus DNA was used as a template for PCRamplification using the following two oligonucleotides:

cck76: 5′-caa tag ggg tca tgg acc c-3′ (SEQ ID NO. 188) cck77: 5′-tcattc tgt gca gag ttg gc-3′ (SEQ ID NO. 189)The resulting 220 bp amplification product was cloned using the TACloning™ Kit (Invitrogen, San Diego, Calif.) and the insert used toscreen the mouse phage library. A positively hybridizing 5.8 kb HindIIIfragment from one of the resultant phage clones was subcloned intopGP1f. The orientation of the insert of this subclone, pHC3′ENfa, issuch that the polylinker XhoI site is adjacent to the 5′ end of theinsert and the SalI site adjacent to the 3′ end. Nucleotide sequenceanalysis of a portion of this HindIII fragment confirmed that itcontained the 3′ heavy chain enhancer. The insert of pHC3′ENfa includesan XhoI site approximately 1.9 kb upstream of the EcoR1 site at the coreof the enhancer sequence. This XhoI site was eliminated by partialdigestion, Klenow fill-in, and religation, to create the clone pH3′Efx,which includes unique XhoI and SalI sites, respectively, at the 5′ and3′ ends of the insert.

The 3′ end of the human γ3/γ1 clone P1-570 was subcloned as follows:P1-570 DNA was digested with NotI, klenow treated, then digested withXhoI; and the 13 kb end fragment isolated and ligated to plasmid vectorpGP2b which had been digested with BamHI, klenow treated, and thendigested with XhoI. The resulting plasmid, pPX-3, has lost thepolylinker NotI site adjacent to the polylinker XhoI site at the 5′ endof the insert; however, the XhoI site remains intact, and the insert canbe isolated by digestion with NotI and XhoI, or SalI and XhoI. The 3′enhancer containing XhoI/SalI insert of pH3′Efx was isolated and ligatedinto the 3′ SalI site of pPX-3 to create the plasmid pHP-3a. Theenhancer containing fragment within the pHP-3a insert is ligated in theopposite orientation as the 3′ end of the P1-570 clone. Therefore,pHP-3a contains an internal SalI site, and the insert is isolated bydigestion with XhoI and NotI. Because this is an enhancer element, 5′ to3′ orientation is generally not critical for function.

HCo7.

To prepare the HCo7 DNA mixture for pronuclear microinjection, DNA fromeach of the five plasmids described above was digested with restrictionenzymes and separated on an agarose gel. Clone pH3V4D was cut with NotI;pCOR1xa was cut with NotI; p11-14 was cut with NotI; pP1-570 was cutwith NotI and SalI; and pHP-3a was cut with NotI and XhoI. The DNAinserts were electroeluted and further purified on an equilibrium CsClgradient without EtBr. The inserts were dialyzed into injection bufferand mixed as follows: 50 microliters of pH3V4D insert @ 20.4ng/microliter; 50 microliters of pCOR1xa insert @ 20.8 ng/microliter; 50microliters of p11-14 insert @ 15.6 ng/microliter; 300 microliters ofpP1-570 insert @ 8.8 ng/microliter; 60 microliters of pHP-3a insert @10.8 ng/microliter; and 1.49 ml injection buffer.

HCo7 Transgenic Animals

The HCo7 DNA mixture was microinjected into the pronuclei of one-halfday old embryos, and the embryos transferred into the oviducts ofpseudopregnant females, as described by Hogan et al. (Manipulating themouse embryo, Cold Spring Harbor laboratories, Cold Spring Harbor N.Y.).

Tail tip DNA was isolated from 202 animals that developed frommicroinjected embryos. Southern blot analysis of this DNA, using a probecomprising human μ and DH sequences, revealed 22 founder animals thathad incorporated at least a portion of the HCo7 transgene. FIG. 81 showsan analysis of the expression of human μ and human γ1 in the serum of 6G0 animals that developed from embryos microinjected with HCo7 DNA.Serum levels of human immunoglobulin proteins were measured by ELISA asdescribed in Lonberg et al. (1994) Nature 368: 856. Four of these sixmice showed evidence of incorporation of the transgene by Southern blotanalysis, and three of these mice expressed both human μ and human γ1proteins in their serum. The single transgenic mouse that did notexpress human immunoglobulin proteins was determined by Southern blotanalysis to contain only a low number of copies of the transgene, and itis possible that the entire transgene was not incorporated, or that thismouse was a genetic mosaic. Two of the founder HCo7 mice, #11952 and#11959, were bred with human κ minilocus (KCo4 line 4436) transgenicmice that were also homozygous for disruptions of the endogenous heavy,and κ light chain loci (Lonberg et al. op.cit), to generate mice thatwere homozygous for the two endogenous locus disruptions and hemizygousfor the two introduced human miniloci, KCo4 and HCo7. Five of theseso-called double-transgenic/double-deletion mice were analyzed forexpression of human IgM, human IgG1, and human IgG3. As a control, threeHC2/KCo4 double-transgenic/double-deletion mice were included in theanalysis. This experiment is presented in FIG. 82. The ELISA data inthis figure was collected as in Lonberg et al. (op.cit), except that fordetection of human IgG3, the coating antibody was a specific mAbdirected against human IgG3 (cat. #08041, Pharmingen, La Jolla, Calif.);the other details of the IgG3 assay were identical to those publishedfor IgG1. While the HC2/KCo4 mice express only human IgM and human IgG1,the HCo7/KCo4 mice also express human IgG3 in addition to these twoisotypes. Expression of human γ3 and γ1 in the HCo7 mice has also beendetected by PCR amplification of cDNA synthesized from RNA isolated fromthe spleen of a transgenic mouse. FIG. 83 depicts PCR amplificationproducts synthesized using spleen cDNA from three different lines oftransgenic mice: line 2550 is an HC2 transgenic line, while lines 11959and 11952 are HCo7 transgenic lines. Single stranded cDNA wassynthesized from spleen RNA as described by Taylor et al. (1992) NucleicAcid Res. 20: 6287. The cDNA was then PCR amplified using the followingtwo oligonucleotides:

o-382: (SEQ ID NO. 190) 5′-gtc cag aat tcg gt(c,g,t)cag ctg gtg(c,g)agtct gg-3′ o-383: (SEQ ID NO. 191) 5′-ggt ttc tcg agg aag agg aag act gacggt cc-3′This primer pair directs the synthesis of PCR products that spans thehinge region of human γ transcripts. Because of differences in thestructures of the human γ1 and γ3 hinge regions, PCR amplificationdistinguishes between these two transcripts. A human γ1 template willdirect the synthesis of a 752 bp PCR product, while human γ3 directs thesynthesis of a 893 bp product. While only human γ1 template isdetectable in the HC2 line 2550 and HCo7 line 11959 spleens, both γ1 andγ3 transcripts are detectable in the HCo7 line 11952 spleen. Because ofthe non-quantitative nature of this assay, and because of differences inγ3 expression between individual animals (shown by ELISA in FIG. 82),the inability to observe γ3 in the HCo7 line 11959 spleen in FIG. 83does not indicate that γ3 is not expressed in this line. Isolated spleencells from the HCo7/KCo4 mice can also be induced to express both IgG1and IgG3 in vitro by stimulation with LPS and IL4. This experiment isshown in FIG. 84. Spleen cells from a 7 week old male HCo7/KCo4double-transgenic/double-deletion mouse (#12496; line 11959/4436) testedfor immunoglobulin secretion in response to the thymus-independent Bcell mitogen, LPS, alone and in conjunction with various cytokines.Splenocytes were enriched for B cells by cytotoxic elimination of Tcells. B-enriched cells were plated in 24 well plates at 2×10⁶ cells perwell in 2 ml of 10% FCS in RPMI-1640. LPS was added to all wells at 10micrograms/ml. IL-2 was-added at 50 units/ml, IL-4 was added at 15ng/ml, IL-6 was added at 15 ng/ml, γIFN was added at 100 units/ml.Cultures were incubated at 37° C., 5% CO₂ for 10 days, then supernatantswere analyzed for human IgG1 and IgG3 by ELISA. All reagents for ELISAwere polyclonal anti-serum from Jackson Immunologicals (West Grove,Pa.), except the capture anti-human IgM, which was a monoclonal antibodyfrom The Binding Site (Birmingham, UK).

Example 38

This example demonstrates the successful introduction into the mousegenome of functional human light chain V segments by co-injection of ahuman κ light chain minilocus and a YAC clone comprising multiple humanV_(κ) segments. The example shows that the V_(κ) segment genes containedon the YAC contribute to the expressed repertoire of human κ chains inthe resultant mouse. The example demonstrates a method for repertoireexpansion of transgene-encoded human immunoglobulin proteins, andspecifically shows how a human κ chain variable region repertoire can beexpanded by co-introduction of unlinked polynucleotides comprising humanimmunoglobulin variable region segments.

Introduction of Functional Human Light Chain V Segments by Co-Injectionof Vk Containing Yeast Artificial Chromosome Clone DNA and k Light ChainMinilocus Clone DNA

I. Analysis of a Yeast Strain Containing Cloned Human Vk Gene Segments.

Total genomic DNA was isolated from a yeast strain containing a 450 kbyeast artificial chromosome (YAC) comprising a portion of the humanV_(κ) locus (ICRF YAC library designation 4x17E1). To determine theidentity of some of the V_(κ) gene segments included in this YAC clone,the genomic DNA was used as a substrate for a series of V_(κ) familyspecific PCR amplification reactions. Four different 5′ primers wereeach paired with a single consensus 3′ primer in four sets ofamplifications. The 5′ primers were: o-270 (5′-gac atc cag ctg acc cagtct cc-3′) (SEQ ID NO. 192), o-271 (5′-gat att cag ctg act cag tctcc-3′) (SEQ ID NO. 193), o-272 (5′-gaa att cag ctg acg cag tct cc-3′)(SEQ ID NO. 194), and o-273 (5′-gaa acg cag ctg acg cag tct cc-3′) (SEQID NO. 195). These primers are used by Marks et al. (Eur. J. Immunol.1991. 21, 985) as V_(κ) family specific primers. The 3′ primer, o-274(5′-gca agc ttc tgt ccc aga ccc act gcc act gaa cc-3′) (SEQ ID NO. 196),is based on a consensus sequence for FR3. Each of the four sets ofprimers directed the amplification of the expected 0.2 kb fragment fromyeast genomic DNA containing the YAC clone 4x17E1. The 4 different setsof amplification products were then gel purified and cloned into thePvuII/HindIII site of the plasmid vector pSP72 (Promega). Nucleotidesequence analysis of 11 resulting clones identified seven distinct Vgenes. These results are presented below in Table 14.

TABLE 14 Identification of human V_(κ) segments on the YAC 4x17E1.identified PCR primers clone # gene Vκ family o-270/o-274 1 L22* I ″ 4L22* I ″ 7 O2* or O12 I o-271/o-274 11 A10* VI ″ 15 A10* VI o-272/o-27420 A4* or A20 I ″ 21 A11* III ″ 22 A11* III ″ 23 A11* III ″ 25 O4* orO14 I o-273/o-274 36 L16* or L2 III *Gene segments mapped within thedistal V_(κ) cluster (Cox et al. Eur. J. Immunol. 1994. 24, 827; Pargentet al. Eur. J. Immunol. 1991. 21, 1829; Schable and Zachau Biol. Chem.Hoppe-Seyler 1993. 374, 1001)

All of the sequences amplified from the YAC clone are eitherunambiguously assigned to V_(κ) genes that are mapped to the distalcluster, or they are compatible with distal gene sequences. As none ofthe sequences could be unambiguously assigned to proximal V genes, itappears that the YAC 4x17E1 includes sequences from the distal Vκregion. Furthermore, one of the identified sequences, clone #7 (VkO2),maps near the J proximal end of the distal cluster, while anothersequence, clones #1 and 4 (VkL22), maps over 300 kb upstream, near the Jdistal end of the distal cluster. Thus, if the 450 kb YAC clone 4x17E1represents a non-deleted copy of the corresponding human genomefragment, it comprises at least 32 different V_(κ) segments. However,some of these are non-functional pseudogenes.

2. Generation of Transgenic Mice Containing YAC Derived V_(κ) GeneSegments.

To obtain purified YAC DNA for microinjection into embryo pronuclei,total genomic DNA was size fractionated on agarose gels. The yeast cellscontaining YAC 4x17E1 were imbedded in agarose prior to lysis, and YACDNA was separated from yeast chromosomal DNA by standard pulse field gelelectrophoresis (per manufacturers specifications: CHEF DR-IIelectrophoresis cell, BIO-RAD Laboratories, Richmond Calif.). Sixindividual pulse field gels were stained with ethidium bromide and theYAC clone containing gel material was cut away from the rest of the gel.The YAC containing gel slices were then imbedded in a new (low meltingtemperature) agarose gel cast in a triangular gel tray. The resultingtriangular gel was extended at the apex with a narrow gel containing twomoles/liter sodium acetate in addition to the standard gel buffer (FIG.85).

The gel was then placed in an electrophoresis chamber immersed instandard gel buffer. The “Y”-shaped gel former rises above the surfaceof the buffer so that current can only flow to the narrow high salt gelslice. A Plexiglas block was placed over the high salt gel slice toprevent diffusion of the NaOAc into the gel buffer. The YAC DNA was thenelectrophoresed out of the original gel slices and into the narrow highsalt block. At the point of transition from the low salt gel to the highsalt gel, there is a resistance drop that effectively halts themigration of the YAC DNA through the gel. This leads to a concentrationof the YAC DNA at the apex of the triangular gel. Followingelectrophoresis and staining, the concentrated YAC DNA was cut away fromthe rest of the DNA and the agarose digested with GELase (EPICENTRETechnologies). Cesium chloride was then added to the YAC DNA containingliquid to obtain a density of 1.68 g/ml. This solution was centrifugedat 37,000 rpm for 36 hrs to separate the DNA from contaminatingmaterial. 0.5 ml fractions of the resulting density gradient wereisolated and the peak DNA containing fraction dialyzed against 5 mM tris(pH 7.4)/5 mM NaCl/0.1 M EDTA. Following dialysis, the concentration ofthe resulting 0.65 ml solution of YAC DNA was found to be 2micrograms/ml. This DNA was mixed with purified DNA insert from plasmidspKC1B and pKV4 (Lonberg et al. 1994. Nature 368, 856) at a ratio of20:1:1 (micrograms YAC4x17E1:KC1B:KV4). The resulting 2 microgram/mlsolution was injected into the pronuclei of half-day mouse embryos, and95 surviving microinjected embryos transferred into the oviducts ofpseudo-pregnant females. Thirty nine mice were born that developed fromthe microinjected embryos. Two of these mice, #9269 and #9272, were usedto establish transgenic lines. The lines are designated KCo5-9269 andKCo5-9272.

A Southern blot analysis of genomic DNA from mice of lines KCo5-9269 andKCo5-9272 was carried out to determine if YAC 4x17E1 derived V_(κ)segments had been incorporated in their genomes. A V_(κ) gene segment,VkA10 (accession #: x12683; Straubinger et al. 1988. Biol. Chem.Hoppe-Seyler 369, 601-607), from the middle of the distal VK cluster waschosen as a probe for the Southern blot analysis. To obtain the clonedprobe, the VkA10 gene was first amplified by PCR. The two oligonucleotides, o-337 (5′-cgg tta aca tag ccc tgg gac gag ac-3′) (SEQ IDNO. 197) and o-338 (5′-ggg tta act cat tgc ctc caa agc ac-3′) (SEQ IDNO. 198), were used as primers to amplify a 1 kb fragment from YAC4×17E1. The amplification product was gel purified, digested withHincII, and cloned into pUC18 to obtain the plasmid p17E1A10. The insertof this plasmid was then used to probe a southern blot of KCo5-9269 andKCo5-9272 DNA. The blot showed hybridization of the probe to theexpected restriction fragments in the KCo5-9272 mouse DNA only. Thisindicates that the VkA10 gene is incorporated into the genome ofKCo5-9272 mice and not KCo5-9269 mice. Line KCo5-9272 mice were thenbred with HC2-2550/JHD/JKD mice to obtain mice homozygous fordisruptions of the endogenous heavy and κ light chain loci, and hemi- orhomozygous for the HC2 and KCo5 transgenes. Animals that are homozygousfor disruptions of the endogenous heavy and k light chain loci, andhemi- or homozygous for human heavy and k light chain transgenes aredesignated double transgenic/double deletion mice.

A CDNA cloning experiment was carried out to determine if any of theYAC-derived V_(κ) genes are expressed in line KCo5-9272 mice. The doubletransgenic/double deletion mouse #12648 (HC2-2550/KCo5-9272/JHD/JKD) wassacrificed and total RNA isolated from the spleen. Single stranded cDNAwas synthesized from the RNA and used as a template in four separate PCRreactions using oligonucleotides o-270, o-271, o-272, and o-273 as 5′primers, and the Ck specific oligonucleotide, o-186 (5′-tag aag gaa ttcagc agg cac aca aca gag gca gtt cca-3′) (SEQ ID NO. 173), as a 3′primer. The amplification products were cloned into the pCRII TA cloningvector (Invitrogen). The nucleotide sequence of 19 inserts wasdetermined. The results of the sequence analysis are summarized in Table15 below.

TABLE 15 Identification of human Vk genes expressed in mouse lineKCo5-9272. identified PCR primers clone # gene Vk family o-270/o-186 1L15* I ″ 3 L18** I ″ 7 L24** I ″ 9 L15* I ″ 10 L15* I o-271/o-186 15A10** VI ″ 17 A10** VI ″ 18 A10** VI ″ 19 A10** VI ″ 21 A10** VIo-272/o-186 101 A27* III ″ 102 L15* I ″ 103 A27* III ″ 104 A27* IIIo-273/o-186 35 A27* III ″ 38 A27* III ″ 44 A27* III ″ 45 A27* III ″ 48A27* III *Vk genes encoded by transgene plasmid sequences. **Vk genesencoded uniquely by YAC derived transgene sequences.

These results show that at least 3 of the YAC derived V, gene segments,A10, L18, and L24, contribute to the expressed human repertoire of theline KCo5-9272 mice.

To determine the effect of this increased repertoire on the size of thevarious B220+ cell populations in the bone marrow and spleen, a flowcytometric analysis was carried out on line KCo5-9272 mice. Part of thisanalysis is shown in FIGS. 86 and 87. Two double transgenic/doubledeletion mice, one containing the KCo5 transgene, and one containing theKCo4 transgene, are compared in this experiment. These two transgenesshare the same joining and constant region sequences, as well as thesame intronic and 3′ enhancer sequences. They also share four differentcloned V gene segments; however, the KCo5 transgene includes theadditional V segments derived from YAC 4x7E1 that are not included inthe KCo4 transgene. Cells were isolated from mouse #13534(HC2-2550/KCo5-9272/JHD/JKD) and mouse #13449(HC2-2550/KCo4-4436/JHD/JKD). Bone marrow cells were stained withanti-mouse B220 (Caltag, South San Francisco, Calif.), anti-mouse CD43(Pharmingen, La Jolla, Calif.), and anti-human IgM (Jackson Immunologic,West Grove, Pa.). Spleen cells were stained with anti-mouse B220 andanti-human IgM.

FIG. 86 shows a comparison of the B cell, and B cell progenitorpopulations in the bone marrow of KCo5 and KCo4 mice. The fraction of Bcells in the bone marrow (B220⁺, IgM⁺) is approximately three timeshigher in the KCo5 mice (6%) than it is in the KCo4 mice (2%). The pre-Bcell population (B220⁺, CD43⁻, IgM⁻) is also higher in the KCo5 mice(9%, compared to 5% for KCo4). Furthermore, the pro-B compartment(B220⁺, CD43⁺) is elevated in these mice (11% for KCo5 and 5% for KCo4).Although each of these three compartments is larger in the KCo5 micethan it is in the KCo4 mice, the levels are still approximately halfthat found in wild type mice. The increase in the number of bone marrowB cells is presumably a direct consequence of the increased repertoiresize. The larger primary repertoire of these mice may provide formembrane Ig with some minimal threshold affinity for endogenousantigens. Receptor ligation could then allow for proliferation of thoseB cells expressing the reactive Ig. However, because the pre-B and pro-Bcells do not express light chain genes, the explanation for theincreased sizes of these two compartments in the KCo5 mice is notimmediately apparent. The B cell progenitor compartments may be largerin KCo5 mice because the increased number of B cells creates a bonemarrow environment that is more conducive to the expansion of thesepopulations. This effect could be mediated directly by secreted factorsor by cell-cell contact between B cells and progenitor cells, or itcould be mediated indirectly, by titration of factors or cells thatwould otherwise inhibit the survival or proliferation of the progenitorcells.

FIG. 87 shows a comparison of the splenic B cell (B220⁺, IgM⁺)populations in KCo5 and KCo4 mice. The major difference between thesetwo mice is the relative sizes of B220^(dull) B cell populations (6% inthe KCo5 mice and 13% in the KCo4 mice). The B220^(dull) cells arelarger than the B₂₂₀ ^(bright) B cells, and a higher fraction of themexpress the 1 light chain. These are characteristics of the so-called B1population that normally dominates the peritoneal B cell population inwild type mice. The spleens of the KCo4 mice comprise an anomalouslyhigh fraction of B220^(dull) cells, while the KCo5 mice have a morenormal distribution these cells. However, both strains containapproximately one-half to one-third the normal number of B cells in thespleen.

Example 39

This example demonstrates the successful use of KCo5 transgenic mice ofExample 38 to isolate hybridoma clones that secrete high affinity,antigen specific, human IgG monoclonal antibodies.

Immunization. A double deletion/double transgenic mouse(KCo5-9272/HC2-2550/JHD/JKD, #12657) was immunized intraperitoneallyevery other week for eight weeks with 4 to 10×10⁶ irradiated T4D3 cells,a murine T cell line expressing human CD4 (Dr. Jane Pames, StanfordUniversity) followed by one injection intraperitoneally two weeks laterof 20 mg soluble recombinant human CD4 (sCD4; Intracell) in incompleteFruend's adjuvant (Sigma). The mouse was boosted once 3 days prior tofusion with 20 mg sCD4 intravenously.

Hybridoma fusion. Single cell suspensions of splenic lymphocytes fromthe immunized mouse were fused to one-sixth the number of P3X63-Ag8.653nonsecreting mouse myeloma cells (ATCC CRL 1580) with 50% PEG (Sigma).Cells were plated at approximately 2×10⁵ in flat bottom microtiterplates, followed by a two week incubation in selective medium containing20% Fetal Clone Serum (HyClone), 18% “653” conditioned medium, 5% Origen(IGEN), 4 mM L-glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 0.055 mM2-mercaptoethanol, 50 units/ml mM penicillin, 50 mg/ml streptomycin, 50mg/ml mM gentamycin and 1×HAT (Sigma; the HAT was added 24 hrs after thefusion). After two weeks, cells were cultured in medium in which the HATwas replaced with HT. Wells were screened by ELISA and flow cytometryonce extensive hybridoma growth or spent medium was observed.

Hybridoma screening by ELISA. To detect anti-CD4 mAbs, microtiter plates(Falcon) were coated overnight at 4° C. with 50 ml of 2.5 mg/ml of sCD4in PBS, blocked at RT for 1 hr with 100 ml of 5% chicken serum in PBS,and then sequentially incubated at RT for 1 hr each with 1:4 dilutionsof supernatant from hybridomas, 1:1000 dilution of F(ab′)₂ fragments ofhorseradish peroxidase (HRPO)-conjugated goat anti-human IgG (Jackson)or 1:250 dilution of HRPO-conjugated goat anti-human Igk antibodies(Sigma) plus 1% normal mouse serum, and finally with 0.22 mg/ml ABTS in0.1 M citrate phosphate buffer, pH 4 with 0.0024% H₂O₂. Plates werewashed 3-6 times with wash buffer (0.5% Tween-20 in PBS) between allincubations, except the first. Diluent (wash buffer with 5% chickenserum) was used to dilute the supernatants and the HRPO conjugates.Absorbance was measured using dual wavelengths (OD at the referencewavelength of 490 nm was subtracted from the OD at 415 nm).

To detect mouse λ-containing mAbs, the above ELISA protocol was used,with the following exceptions. Wells of microtiter plates were coatedwith 100 ml of 1) 1.25 mg/ml goat anti-mouse λ (Pierce), 2) 1.25 mg/mlgoat anti-human Fcγ (Jackson), or 3) 2.5 mg/ml sCD4 (ABT). For thedetection step, 100 ml of 1:5000 goat anti-mouse 1 (SBA) conjugated tobiotin was used followed by 100 ml of 1:1000 streptavidin conjugated toHRPO (Jackson). Murine and human mAb standards were used at theindicated concentrations. To look for cross-reactivity to unrelatedantigens, wells were coated with CEA (Crystal Chem), KLH (CalBiochem),HSA (Sigma), BSA (Sigma) or OVA (Sigma; all at 2 mg/ml, except CEA whichwas at 2.5). Appropriate antibodies were titered and used as positivecontrols (human IgM anti-CEA (GenPharm), rabbit anti-KLH (Sigma), sheepanti-HSA (The Binding Site), sheep anti-BSA (The Binding Site), andsheep anti-OVA (The Binding Site)). Any bound antibody was detected withHRPO conjugates of goat anti-human IgM, donkey anti-rabbit IgG or donkeyanti-sheep IgG (all diluted 1:1000 and obtained from Jackson).Otherwise, the standard ELISA protocol was followed.

Hybridoma screening by flow cytometric assay. To further screen for mAbsreactive with native cell-surface CD4, 5×10⁵ SupT1 cells (ATCC CRL 1942)were incubated on ice with a 1:2 dilution of spent supernatant from thefusion plates for 30 min, washed twice with cold stain buffer (0.1% BSA,0.02% NaN₃ in PBS), incubated with 1.5 mg/ml of an F(ab′)₂ fragment ofFITC-conjugated goat anti-human Fcg (FITC-GaHuIgG; Jackson) for 15 min,washed once and analyzed immediately on a FACScan (Becton-Dickinson).

CD4 reactive hybridomas. Using the ELISA and flow cytometric techniquesdescribed above, 12 hybridoma clones were identified that secreted humanIgG specifically reactive with native human CD4. Ten of these twelveclones were further subcloned. Eight of these subclones were identifiedas human IgG1_(κ) secreting hybridomas. The other two expressed a mouse80 light chain. The parent wells for the 8 fully human clones were:1E11, 2E4, 4D1, 6C1, 6G5, 7G2, 10C5, and 1G1. Flow cytometric assays ofthe binding of 3 of the fully human IgGk subclones (4D1.4, 6G5.1, and10C5.6) are shown in FIG. 88.

FIG. 88 shows binding of IgG_(κ) anti-nCD4 monoclonal antibodies to CD4+SupT1 cells. Cells from log phase growth cultures were washed andstained with no monoclonal antibody, 4E4.2 (as a negative control),chimeric Leu3a (as a positive control), or with one of the 10 human IgGanti-nCD4 monoclonal antibodies. Any bound monoclonal antibody wasdetected with FITC-conjugated goat anti-human Fcγ. All ten monoclonalantibodies bound to SupT1 cells, although data is shown here for onlythree of them.

Analysis of human antibody secretion by cloned hybridomas. To comparethe growth and secretion levels of mAbs, the subclones were put intoreplicate cultures in HT medium in 24 well plates at an initial densityof 2×10⁵ cells/ml. Each day for 7 days, one of the replicate culturesfor each subclone was harvested and cell numbers, cell viability (byTrypan blue exclusion) and the amount of mAb in the supernatant (by aquantitative ELISA for total human γ) were determined. Table 16 showsdata for antibody secretion by 7 of the hybridoma subclones.

TABLE 16 Secretion Levels For Human IgGk Anti-nCD4 Monoclonal AntibodiesSubclone pg/cell pg/cell/d 1E11.15 3.9 0.56 1G1.9 11 1.5 4D1.4 1.4 0.916C1.10 3.3 0.48 6G5.1 7.8 1.1 7G2.2 4.4 0.63 10C5.6 8.0 1.1 *pg/cell =(maximum amount of mAb)/(maximum number of viable cells) pg/cell/d =(pg/cell)/7 days

Purification of human mAbs. The individual hybridoma clones were grownin medium without HT and Origen and the FCS was gradually decreased toapproximately 2-3% in the final 11 cultures. Supernatants were harvestedonce the viability of the hybridomas fell below approximately 30%. Topurify the IgGk mAbs, the spent supernatants were centrifuged to removecells, concentrated via ultrafiltration to approximately 50 to 100 mls,diluted 1:5 with PBS, pH 7.4 and loaded onto a 5 ml Protein A(Pharmacia) column. After washing with 3-5 column volumes of PBS, thehuman IgGk mAbs were eluted with 0.1HC1, 150 mM NaCl, pH 2.8 andimmediately neutralized with 1M Tris base. Column fractions containingmaterial with an OD₂₈₀>0.2 were pooled and dialyzed into PBS. The OD₂₈₀was then determined and an absorbtivity coefficient of 1.4 was used tocalculate the protein concentration of the human IgG. No mAb wasdetected in the flow through and the % recoveries ranged from 93 to100%. Three to six mgs of each purified mAb were obtained, with >90%purity.

Analysis of monoclonal antibodies from cloned hybridomas. To investigatethe specificity of binding of mAbs, human PBMC were isolated over Ficolland stained as follows. Human PBMC (106) in stain buffer were incubatedfor 30 min on ice, in separate reactions, with equal volumes ofsupernatant from each of three of the subcloned hybridomas (4D1.4,6G5.1, and 10C5.6), or with an isotype matched negative control mAb,washed twice, and incubated 20 min on ice with 1 mg/ml of FITC-GaHuIgGalong with either 10 ml of mouse anti-human CD4 mAb (Leu3a;Becton-Dickinson) conjugated to phycoerythrin (PE), 10 ml of mouseanti-human CD8 mAb (Leu2a; Becton-Dickinson) conjugated to PE, or 5 mlof mouse anti-human CD19 mAb (SJ25-C1; Caltag) conjugated to PE. Gatedlymphocytes were then analyzed on a FACScan flow cytometer (BectonDickinson, San Jose, Calif.). All three of the antibodies were found tobind specifically to the CD4 fraction of the human PBMC.

To approximate the location of the epitope recognized by these threemAbs, 5×10⁵ SupT1 cells were pre-incubated for 20 min on ice withbuffer, 2.5 mg/ml RPA-T4, or 2.5 mg/ml Leu3a in stain buffer, then for30 min with one of the 10 human IgG mAbs (in supernatant diluted 1:2)and finally with 0.5 mg/ml FITC-conjugated goat anti-human Fcγ to detectany bound human IgG. Cells were washed twice with stain buffer prior toand once after the last step. The results of this blocking assay areshown in FIG. 89. None of the three antibodies share an epitope withRPA-T4, while 6G5.1 and 10C5.6 appear to recognize the same (or anadjacent) epitope as that recognized by Leu3a.

Rate and Equilibrium Constant Determinations.

Human sCD4 (2500 to 4200 RU) was immobilized by covalent couplingthrough amine groups to the sensor chip surface according tomanufacturer's instructions. Antibody dilutions were flowed over theantigen-coupled sensor chips until equilibrium was reached, and thenbuffer only was allowed to flow. For each phase of the reaction, bindingand dissociation, the fraction of bound antibody was plotted over time.The derivative of the binding curve (dR/dt) was calculated and plottedagainst the response for each concentration. To calculate theassociation rate constant (K_(assoc)), the slopes of those resultinglines were then plotted against the concentration of the monoclonalantibody. The slope of the line from this graph corresponded to thek_(asoc). The dissociation rate constant (k_(dissoc)) was calculatedfrom the log of the drop in response (during the buffer flow phase)against the time interval. The Ka was derived by dividing the k_(assoc)by the k_(dissoc). The measured rate and affinity constant data for 5different purified monoclonal antibodies derived from the KCo5/HC2double transgenic/double deletion mice, and one purified antibodyobtained from a commercial source (Becton Dickinson, San Jose, Calif.),is presented in Table 17.

TABLE 17 Rate and affinity constants for monoclonal antibodies that bindto human CD4. Antibody Source k_(ass) (M⁻¹s⁻¹) k_(diss) (s⁻¹) Ka (M⁻¹)human HC2/KCo5 2.7 × 10⁵ 4.6 × 10⁻⁵ 5.8 × 10⁹ IgG1k transgenic humanHC2/KCo5 9.1 × 10⁴ 2.2 × 10⁻⁵ 4.2 × 10⁹ IgG1k transgenic human HC2/KCo59.8 × 10⁴ 4.2 × 10⁻⁵ 2.3 × 10⁹ IgG1k transgenic human HC2/KCo5 1.1 × 10⁵1.0 × 10⁻⁵  1.1 × 10¹⁰ IgG1k transgenic k_(ass) Hybridoma AntibodySource (M⁻¹s⁻¹) k_(diss) (s⁻¹) Ka (M⁻¹) 10C5.6 human HC2/ 7.4 × 10⁴ 1.6× 10⁻⁵ 4.5 × 10⁹  IgG1k KCo5 transgenic Leu3a mouse Becton 1.5 × 10⁵ 4.2× 10⁻⁶ 3.7 × 10¹⁰ IgG1k Dickinson

Mixed Lymphocyte Reaction (MLR). To compare the in vitro efficacy of thehuman monoclonal antibody 10C5.6, derived from the KCo5 transgenicmouse, to that of the mouse antibody Leu3a, an MLR assay was performed.Human PBMC from 2 unrelated donors were isolated over Ficoll and CD4+PBL from each donor-were purified using a CD4 column (Human CD4 Cellect,Biotex Laboratories, Inc., Canada) according to manufacturer'sdirections. Inactivated stimulator cells were obtained by treating PBMCfrom both donors with 100 mg/ml mitomycin C (Aldrich) in culture medium(RPMI 1640 with 10% heat-inactivated human AB serum (from NABI), Hepes,sodium pyruvate, glutamine, pen/strep and b-mercaptoethanol (all used atmanufacturer's recommended concentrations)) for 30 min at 37° C.followed by 3 washes with culture medium. Varying concentrations of mAbsdiluted in culture medium or culture medium only were sterile filteredand added at 100 ml per well in triplicate in a 96 well round bottomplate. Fifty ml of 10⁵ CD4+ PBL from one donor in culture medium and 105mitomycin C-treated PBMC from the other donor in 50 ml of culture mediumwere then added to each well. Control plates with CD4+ PBL respondersalone plus mabs were set up to control for any toxic or mitogeniceffects of the mAbs. A stimulator only control and a media backgroundcontrol were also included. After seven days in a 37° C., 5% CO₂humidified incubator, 100 ml of supernatant from each well was removedand 20 ml of colorimetric reagent (Cell Titer 96AQ kit, PromegaCorporation, Madison, Wis.) was added. Color was allowed to develop for4 to 6 hrs and plates were read at 490 μm. The results of thisexperiment, depicted in FIG. 90, show that the human IgG1k antibody10C5.6 is at least as effective as Leu3a at blocking the function ofhuman PBMC CD4 cells in this assay.

Example 40 Binding Characteristics of Human IqGkappa Anti-CD4 MonoclonalAntibodies

This example provides the binding characteristics of human IgG_(κ)monoclonal antibodies derived from hybridoma clones obtained fromHC2/KCo5/JHD/JCKD transgenic mice immunized with human CD4. Themonoclonal antibodies are shown to have high avidity and affinity forrecombinant and natural human CD4.

Cells from 10 individual hybridoma cell lines (1E11, 1G2, 6G5, 10C5,1G1, 6C1, 2E4, 7G2, 1 F8 and 4D1) that secrete human IgG kappamonoclonal antibodies (mAB) reactive with human CD4, were derived fromJHD/JCKD/HC2/KCo5 transgenic mice. The cell lines were grown in culture,and antibody proteins were isolated from the supernatant (Fishwild, etal. 1996, Nature Biotechnology 14, 845-851, which is incorporated hereinby reference). Antibody purified by Protein A affinity chromatographywas used to measure binding constants. The results are displayed inTables 18 and 19.

The rate and equilibrium constants presented in Table 18 were determinedwith a BIAcore (Pharmacia Biosensor) using goat anti-human IgG(Fc-specific) coupled to the sensor chip and flowing a saturatingconcentration of mAb over followed by various concentrations of antigen(rCD4). These constants were derived from three experiments usingpurified mAbs.

TABLE 18 Affinity and Rate Constants. Rate Constants (mean ± SD) HumanmAb k_(assoc) (M⁻¹s⁻¹) k_(dissoc) (s⁻¹) K_(a) (M⁻¹) 1E11.15 1.7 (±0.15)× 10⁵ 3.5 (±0.09) × 10⁻³ 5.0 × 10⁷ 6C1.10 1.8 (±0.44) × 10⁵ 3.3 (±0.04)× 10⁻³ 5.4 × 10⁷ 1G1.9 1.2 (±0.18) × 10⁵ 9.4 (±0.22) × 10⁻⁴ 1.3 × 10⁸6G5.1  9.3 (±1.1) × 10⁴ 6.9 (±0.36) × 10⁻⁴ 1.4 × 10⁸ 10C5.6 9.4 (±0.98)× 10⁴ 7.1 (±0.36) × 10⁻⁴ 1.3 × 10⁸ 2E4.2 1.8 (±0.10) × 10⁵ 2.5 (±0.05) ×10⁻³ 7.1 × 10⁷ 4D1.4 2.5 (±0.55) × 10⁵ 3.4 (±0.15) × 10⁻³ 7.3 × 10⁷7G2.2 2.4 (±0.31) × 10⁵ 3.3 (±0.07) × 10⁻³ 7.3 × 10⁷ 1F8.3 1.8 (±0.24) ×10⁵ 4.3 (±0.14) × 10⁻³ 4.3 × 10⁷ 1G2.10 2.2 (±0.26) × 10⁵ 2.3 (±0.03) ×10⁻³ 9.8 × 10⁷ chi Leu3a 1.5 (±0.35) × 10⁵ 2.3 (±0.12) × 10⁻⁴ 6.6 × 10⁸

The rate and equilibrium constants presented in Table 19 were determinedwith a BIAcore, using antigen (rCD4) coupled to the sensor chip andflowing mAb over. These constants were derived from at least threeindependent experiments using purified mAbs.

TABLE 19 Avidity and Rate Constants Rate Constants (mean ± SD) Human mAbk_(assoc) (M⁻¹s⁻¹) k_(dissoc) (s⁻¹) K_(a) (M⁻¹) 1E11.15 2.8 (±0.22) ×10⁵ 4.5 (±0.43) × 10⁻⁵ 6.2 × 10⁹ 6C1.10 2.0 (±0.25) × 10⁵ 4.0 (±0.63) ×10⁻⁵ 5.1 × 10⁹ 1G1.9 9.1 (±0.95) × 10⁴ 2.2 (±0.71) × 10⁻⁵ 4.2 × 10⁹6G5.1 1.1 (±0.41) × 10⁵ 1.0 (±0.34) × 10⁻⁵ 1.1 × 10¹⁰ 10C5.6  7.4 (±1.5)× 10⁴ 1.6 (±0.57) × 10⁻⁵ 4.5 × 10⁹ 2E4.2 1.4 (±0.15) × 10⁵ 2.2 (±0.25) ×10⁻⁵ 6.3 × 10⁹ 4D1.4 9.8 (±0.69) × 10⁴  4.2 (±1.3) × 10⁻⁵ 2.3 × 10⁹7G2.2 1.7 (±0.20) × 10⁵ 5.0 (±0.42) × 10⁻⁵ 3.4 × 10⁹ 1F8.2 1.7 (±0.13) ×10⁵  9.7 (±1.2) × 10⁻⁵ 1.7 × 10⁹ 1G2.10 1.7 (±0.04) × 10⁵ 6.3 (±0.49) ×10⁻⁵ 2.7 × 10⁹ chi Leu3a 4.0 (±0.45) × 10⁵ 1.2 (±0.25) × 10⁻⁵ 3.4 × 10¹⁰Leu3a 1.5 (±0.30) × 10⁵ 4.2 (±0.49) × 10⁻⁶ 3.7 × 10¹⁰

Table 20 provides equilibrium constants for anti-CD4 mABs presented inthe scientific literature.

TABLE 20 Avidity and Rate Constants Reported for Anti- CD4 monoclonalantibodies. Rate Constants (mean ± SD) Human mAb k_(assoc) (M⁻¹s⁻¹)k_(dissoc) (s⁻¹) K_(a) (M⁻¹) CE9.1⁽⁴⁾ NR* NR 3.1 × 10¹⁰ cMT412⁽¹⁾ NR NR5.0 × 10⁹ chi Leu3a⁽²⁾ NR NR 1.0 × 10¹¹ BL4⁽³⁾ NR NR 5.5 × 10⁷ BB14⁽³⁾NR NR 3.3 × 10⁸ cA2⁽⁵⁾ NR NR 1.8 × 10⁹ CDP571⁽⁶⁾ NR NR 7.1 × 10⁹ *NR =not reported ⁽¹⁾J. Cell. Biol. 15E: A179. ⁽²⁾J. Immunol. 145: 2839.⁽³⁾Clin. Immunol. Immunopath. 64: 248. ⁽⁴⁾Biotechnology. 10: 1455.⁽⁵⁾Mol. Immunol. 30: 1443. ⁽⁶⁾European Patent Appl. #0626389A1.

The avidity and affinity determinations described above were performedwith recombinant CD4 (rCD4). To determine the avidity of the humanmonoclonal antibodies for native CD4 (nCD4). An additional binding assaywas used that does not require the antibody to be modified.Specifically, serial dilutions of antibody were incubated with SupT1cells for 6 hr on ice, washed and detected any bound antibody withFITC-goat anti-human Fcγ. The Ka is determined from the concentration ofantibody that gives one-half of the maximum fluorescence (a fourparameter fit was used). The results demonstrate that all ten humanmonoclonal antibodies bind very well to nCD4, with Ka values >10⁹ M⁻¹(Table 21). Most antibodies, including chimeric Leu3a, bound less wellto nCD4 than to rCD4. This could be due to differences in antigendensity as well as to differences between the two antigens.

TABLE 21 Avidity Constants Determined by Flow Cytometry. Ka values (M−1)Ratio of Ka Human mAb rCD4 nCD4* (rCD4/nCD4) 1E11.15 6.2 × 10⁹ 3.3 × 10⁹1.9 6C1.10 5.1 × 10⁹ 3.1 × 10⁹ 1.6 1G1.9 4.2 × 10⁹ 2.3 × 10⁹ 1.9 6G5.11.1 × 10¹⁰ 1.9 × 10⁹ 5.9 10C5.6 4.5 × 10⁹ 1.8 × 10⁹ 2.5 2E4.2 6.3 × 10⁹1.1 × 10⁹ 5.8 4D1.4 2.3 × 10⁹ 2.0 × 10⁹ 1.2 7G2.2 3.4 × 10⁹ 3.3 × 10⁹1.0 1F8.2 1.7 × 10⁹ 3.2 × 10⁹ 0.5 1G2.10 2.7 × 10⁹ 1.9 × 10⁹ 1.4 chiLeu3a 3.4 × 10¹⁰ 5.6 × 10⁹ 6.1 *Human monoclonal antibodies wereincubated in serial dilutions with SupT1 cells for 6 hrs, washed twiceand incubated with FITC-conjugated goat anti-human Fcγ antisera, washedand fixed. The Ka was calculated from the concentration of antibodyyielding one-half of the maximum fluorescence as determined from afour-parameter fit.

Example 41 Identification of Nucleotide Sequences Encoding HumanIqGkappa Anti-CD4 Antibodies

This example demonstrates that a each of the hybridomas tested producesonly one functional heavy or light chain RNA transcript, consistent withproper functioning allelic exclusion. In addition, sequence analysis ofheavy and light chain CDR segments indicates that somatic mutation ofthe immunoglobulin transgenes has taken place.

Cells from five individual hybridoma cell lines (1E11, 1G2, 6G5, 10C5,and 4D1) that secrete human IgG kappa monoclonal antibodies reactivewith human CD4, and derived from JHD/JCKD/HC2/KCo5 transgenic mice, wereused to isolate RNA encoding each of the individual antibodies (Fishwildet al. 1996, Nature Biotechnology 14, 845-851). The RNA was used as asubstrate to synthesize cDNA, which was then used to amplify human Iggamma and kappa transcript sequences by PCR using primers specific forhuman VH, Vkappa, Cgamma, and Ckappa (Taylor et al. 1992, Nucleic AcidsRes. 20, 6287-6295; Larrick, J. W., et al. (1989), Bio/Technology. 7.934-938; Marks, J. D., et al. (1991). Eur. J. Immunol. 21. 985-991;Taylor, et al; 1994, Int. Immunol. 6, 579-591). The amplified Ig heavyand kappa light chain sequences were cloned into bacterial plasmids andnucleotide sequences determined. Analysis of the sequences spanning theheavy chain VDJ and light chain VJ junctions revealed in-frame heavy andlight chain transcripts for each of the 5 clones, and in some casesadditional out-of-frame sterile transcripts representing non-functionalalleles. Consistent with proper functioning allelic exclusion, in nocase was there more than one unique functional heavy or light chaintranscript identified for each of the individual clones. Partialnucleotide sequences for each of the ten functional transcripts areassigned the following sequence I.D. No's: 1E11 gamma [SEQ. ID NO. 199];1E11 kappa [SEQ. ID NO. 200]; 1G2 gamma [SEQ. ID NO. 201]; 1G2 kappa[SEQ. ID NO. 202]; 6G5 gamma [SEQ. ID NO.203]; 6G5 kappa [SEQ. IDNO.204]; 10C5 gamma [SEQ. ID NO. 205]; 10C5 kappa [SEQ. ID NO.206]; 4D1gamma [SEQ. ID NO. 207]; 4D1 kappa [SEQ. ID NO. 208] and are presentedin Table 22. All sequences are presented in a 5′ to 3′ orientation.

TABLE 22 Partial Nucleotide Sequence for Functional Transcripts 1E11gamma (SEQ ID NO: 199)TGCACAAGAACATGAAACACCTGTGGTTCTTCCTCCTCCTGGTGGCAGCTCCCAGATGGGTCCTGTCCCAGGTGCAGCTTCATCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTTCTGGAGCTGGATCCGCCAGCCCCCAGGGAGGGGGCTGGAGTGGATTGGGGAAATCCATCATCGTGGAAGCACCAACTACAACCCGTCCCTCGAGAGTCGAGTCACCCTATCAGTAGACACGTCCAAAAACCAGTTCTCCCTGAGGCTGAGTTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGAGACATTACTATGGTTCGGGGAGTACCTCACTGGGGCCAGGG AACCCTGGTCACC 1E11kappa (SEQ ID NO: 200)GACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCCCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGAACTGTGGCGGCACCATCTGTCTTCATCTT CCC 1G2 gamma (SEQ IDNO: 201) TCCACCATCATGGGGTCAACCGCCATCCTCGCCCTCCTCCTGGCTGTTCTCCAAGGAGTCTGTGCCGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAGTTACTGGATCGCCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCGATCCTGCTGACTCTGATACCAGATACAACCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGTACCGCCTATTTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACCAGCGAACTGGAACTGGTACTTCGTTCTCTGGGGCCGTG GCACCCTGGTCACT 1G2kappa (SEQ ID NO: 202)GACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCAACAGTTTATTAGTTACCCTCAGCTCACTTTCGGCGGAGGGACCAGGGTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCAT CTTCCC 6G5 gamma (SEQID NO: 203) TGCACAAGAACATGAAACACCTGTGGTTCTTCCTCCTCCTGGTGGCAGCTCCCAGATGGGTCCTGTCCCAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGTAAGGGGCTGGAGTGGATTGGGGAAATCAATCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTCGACACGTCCAAGAACCAGTTCTCCCTGAAACTGAGCTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGAGTAATTAATTGGTTCGACCCCTGGGGCCAGGGAACCCTGGT CACC 6G5 kappa (SEQID NO: 204) GACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAATAGTTTCCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTT CCC 10C5 gamma (SEQID NO: 205) ATGAAACACCTGTGGTTCTTCCTCCTCCTGGTGGCAGCTCCCAGATGGGTCCTGTCCCAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGTAAGGGGCTGGAGTGGATTGGGGAAATCAATCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTCGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAGAGTAATTAATTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCT CAG 10C5 kappa (SEQID NO: 206) ATGGACATGATGGTCCCCGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGTTCCCAGGTTCCAGATGCGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGATATTAGCAGCTGGTTAGCCTGGTATCAGCATAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAATAGTTTCCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAAC 4D1 gamma (SEQ ID NO: 207)ATGGGGTCAACCGCCATCCTCGCCCTCCTCCTGGCTGTTCTCCAAGGAGTCTGTGCCGAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCGGCTACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCACATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGAGACCAACTGGGCCTCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCC TCCAAGAAGCTT 4D1kappa (SEQ ID NO: 208)ATGGACATGGAGTTCCCCGTTCAGCTCCTGGGGCTCCTGCTGCTCTGTTTCCCAGGTGCCAGATGTGACATCCAGATGACCCAGTCTCCATCCTCACTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGAGAAAGCCCCTAAGTCCCTGATCTATTCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGCCAACAGTATGATAGTTACCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAAGCTT

Analysis of these DNA sequences demonstrates that the 5 hybridoma clonesrepresent descendants of 4 individual primary B cells. Table 23 showsthe amino acid sequences derived for each of the ten CDR3 regions, andthe assignments for germline gene segments incorporated into each of thegenes encoding these transcripts. The germline assignments are based onpublished gene sequences available from the National Center forBiotechnology Information, National Library of Medicine, NationalInstitutes of Health, Bethesda, Md. Also see: Cook et al. 1994, NatureGenet. 7, 162-168; Tomlinson et al. 1992, J. Mol. Biol. 227, 776-798;Matsuda et al. 1993, Nature Genet. 3, 88-94; Schable and Zachau, 1993,Biol. Chem. Hoppe-Seyler 374, 1001-1022; Cox et al. 1994, Eur. J.Immunol. 24, 827-836; Ravetch et al. 1981, Cell 27, 583-591; Ichihara etal. 1988, EMBO J. 7, 4141-4150; Yamada et al. 1991, J. Exp. Med. 173,395-407; Sanz, 1991, J. Immunol. 147, 1720-1729.

TABLE 23 Germline V(D)J Segment Usage in Hybridoma Transcripts. cloneh.c. CDR3 VH DH JH l.c. CDR3 Vk Jk  1E11 DITMVRGVPH VH4-34 DXP′1 JH4QQYGSSPLT VkA27/ Jk4 (Seq. I.D. No. _) (Seq. I.D. No. _) A11  1G2PANWNWYFVL VH5-51 DHQ52 JH2 QQFISYPQLT VkL18 Jk4 (Seq. I.D. No. _) (Seq.I.D. No. _)  6G5 VINWFDP VH4-34 n.d. JH5 QQANSFPYT VkL19 Jk2 (Seq. I.D.No. _) (Seq. I.D. No. _) 10C5 VINWFDP VH4-34 n.d. JH5 QQANSFPYT VkL19Jk2  4D1 DQLGLFDY VH5-51 DHQ52 JH4 QQYDSYPYT VkL15 Jk2 (Seq. I.D. No. _)(Seq. I.D. No. _) n.d. could not be determined from nucleotide sequence.

Example 42 Construction of Miniqenes for Expression of Human IqGkappaAntiCD4 Antibodies in Transfected Cell Lines

This example demonstrates the process of making a wholly artificial genethat encodes an immunoglobulin polypeptide (i.e., an immunoglobulinheavy chain or light chain). Plasmids were constructed so that PCRamplified V heavy and V light chain cDNA sequences could be used toreconstruct complete heavy and light chain minigenes.

The kappa light chain plasmid, pCK7-96, includes the kappa constantregion and polyadenylation site [SEQ. ID NO. 217], such that-kappasequences amplified with 5′ primers that include HindIII sites upstreamof the initiator methionine can be digested with HindIII and BbsI, andcloned into pCK7-96 digested with HindIII and BbsI to reconstruct acomplete light chain coding sequence together with a polyadenylationsite. This cassette can be isolated as a HindIII/NotI fragment andligated to transcription promoter sequences to create a functionalminigene for transfection into cells.

The gammal heavy chain plasmid, pCG7-96, includes the human gammalconstant region and polyadenylation site [SEQ. ID NO. 218], such thatgamma sequences amplified with 5′ primers that include HindIII sitesupstream of the initiator methionine can be digested with HindIII andAgeI, and cloned into pCG7-96 digested with HindIII and AgeI toreconstruct a complete gammal heavy chain coding sequence together witha polyadenylation site. This cassette can be isolated as a HindIII/SalIfragment and ligated to transcription promoter sequences to create afunctional minigene for transfection into cells.

The following example demonstrates how nucleotide sequence data fromhybridomas can be used to reconstruct functional Ig heavy and lightchain minigenes. The nucleotide sequences of heavy and light chaintranscripts from hybridomas 6G5 and 10C5 were used to design anoverlapping set of synthetic oligonucleotides to create synthetic Vsequences with identical amino acid coding capacities as the naturalsequences. The synthetic heavy and kappa light chain sequences(designated HC6G5 [SEQ. ID NO.219] and LC6G5 [SEQ. ID NO. 220] differedfrom the natural sequences in three ways: strings of repeated nucleotidebases were interrupted to facilitate oligonucleotide synthesis and PCRamplification; optimal translation initiation sites were incorporatedaccording to Kozak's rules (Kozak, 1991, J. Biol. Chem. 266,19867-19870); and, HindIII sites were engineered upstream of thetranslation initiation sites.

A. Synthetic Kappa Light Chain.

Light Chain PCR Reaction 1.

The following oligonucleotides were pooled: o-548 [SEQ. ID NO. 221],o-549 [SEQ. ID NO. 222], o-550 [SEQ. ID NO. 223], o-551 [SEQ. ID NO.224], o-552 [SEQ. ID NO. 225], o-563 [SEQ. ID NO. 226], o-564 [SEQ. IDNO. 227], o-565 [SEQ. ID NO. 228], o-566 [SEQ. ID NO. 229], o-567 [SEQ.ID NO. 230], and amplified with the following 2 primers: o-527 [Seq.I.D. No. 231] and o-562 [SEQ. ID NO. 232].

Light Chain PCR Reaction 2.

The following oligonucleotides were pooled: o-553 [Seq. I.D. No. 233],o-554 [Seq. I.D. No. 234], o-555 [Seq. I.D. No. 235], o-556 [Seq. I.D.No. 236], o-557 [Seq. I.D. No. 237], o-558 [Seq. I.D. No. 238], o-559[Seq. I.D. No. 239], o-560 [Seq. I.D. No. 240], o-561 [Seq. I.D. No.241], o-562 [Seq. I.D. No. 242], and amplified with the following 2primers: o-552 [Seq. I.D. No. 225] and o-493 [Seq. I.D. No. 242].

Light Chain PCR Reaction 3.

The products of light chain PCR reactions 1 and 2 were then combined andamplified with the following two primers: o-493 [Seq. I.D. No. 242] ando-527 [Seq. I.D. No. 231].

The product of light chain PCR reaction 3 was then digested with HindIIIand BbsI and cloned into HindIII/BbsI digested pCK7-96 [Seq. I.D. No.217] to generate pLC6G5 [Seq. I.D. No. 243].

B. Synthetic Gamma Heavy Chain.

Heavy Chain PCR Reaction 1.

The following oligonucleotides were pooled: o-528 [Seq. I.D. No. 244],o-529 [Seq. I.D. No. 245], o-530 [Seq. I.D. No. 246], o-531 [Seq. I.D.No. 247], o-532 [Seq. I.D. No. 248], o-543 [Seq. I.D. No. 249], o-544[Seq. I.D. No. 250], o-545 [Seq. I.D. No. 251], o-546 [Seq. I.D. No.252], o-547 [Seq. I.D. No. 253], and amplified with the following 2primers: o-496 [Seq. I.D. No. 254] and o-542 [Seq. I.D. No. 255].

Heavy Chain PCR Reaction 2.

The following oligonucleotides were pooled: o-533 [Seq. I.D. No. 256],o-534 [Seq. I.D. No. 257], o-535 [Seq. I.D. No. 258], o-536 [Seq. I.D.No. 259], o-537 [Seq. I.D. No. 260], o-538 [Seq. I.D. No. 261], o-539[Seq. I.D. No. 262], o-540 [Seq. I.D. No. 263], o-541 [Seq. I.D. No.264], o-542 [Seq. I.D. No. 255], together with the isolated 439 bp BbsIfragment of pCG7-96 [Seq. I.D. No. 218] and amplified with the following2 primers: o-490 [Seq. I.D. No. 265] and o-520 [Seq. I.D. No. 266].

Heavy Chain PCR Reaction 3.

The products of heavy chain reactions 1 and 2 were then combined andamplified with the following two primers: o-520 [Seq. I.D. No. 266] ando-521 [Seq. I.D. No. 267].

The product of heavy chain reaction 3 was then digested with HindIII andAgeI and cloned into HindIII/AgeI digested pCG7-96 [Seq. I.D. No. 218]to generate pHC6G5 [Seq. I.D. No. 268].

TABLE 24 Primers, Vectors and Products Used in Minigene ConstructionpCK7-96 (SEQ ID NO: 217)TCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGCCAAGCTAGCGGCCGCGGTCCAACCACCAATCTCAAAGCTTGGTACCCGGGAGCCTGTTATCCCAGCACAGTCCTGGAAGAGGCACAGGGGAAATAAAAGCGGACGGAGGCTTTCCTTGACTCAGCCGCTGCCTGGTCTTCTTCAGACCTGTTCTGAATTCTAAACTCTGAGGGGGTCGGATGACGTGGCCATTCTTTGCCTAAAGCATTGAGTTTACTGCAAGGTCAGAAAAGCATGCAAAGCCCTCAGAATGGCTGCAAAGAGCTCCAACAAAACAATTTAGAACTTTATTAAGGAATAGGGGGAAGCTAGGAAGAAACTCAAAACATCAAGATTTTAAATACGCTTCTTGGTCTCCTTGCTATAATTATCTGGGATAAGCATGCTGTTTTCTGTCTGTCCCTAACATGCCCTGTGATTATCCGCAAACAACACACCCAAGGGCAGAACTTTGTTACTTAAACACCATCCTGTTTGCTTCTTTCCTCAGGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGAGGGAGAAGTGCCCCCACCTGCTCCTCAGTTCCAGCCTGACCCCCTCCCATCCTTTGGCCTCTGACCCTTTTTCCACAGGGGACCTACCCCTATTGCGGTCCTCCAGCTCATCTTTCACCTCACCCCCCTCCTCCTCCTTGGCTTTAATTATGCTAATGTTGGAGGAGAATGAATAAATAAAGTGAATCTTTGCACCTGTGGTTTCTCTCTTTCCTCAATTTAATAATTATTATCTGTTGTTTACCAACTACTCAATTTCTCTTATAAGGGACTAAATATGTAGTCATCCTAAGGCGCATAACCATTTATAAAAATCATCCTTCATTCTATTTTACCCTATCATCCTCTGCAAGACAGTCCTCCCTCAAACCCACAAGCCTTCTGTCCTCACAGTCCCCTGGGCCATGGATCCTCACATCCCAATCCGCGGCCGCAATTCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGC PCG7-96 (SEQ ID NO: 218)GAACTCGAGCAGCTGAAGCTTTCTGGGGCAGGCCAGGCCTGACCTTGGCT TTGGGGCAGGGAGGGGGCTAAGGTGAGGCAGGTGGCGCCAGCCAGGTGCACACCCAATGCCCATGAGCCCAGACACTGGACGCTGAACCTCGCGGACAGTTAAGAACCCAGGGGCCTCTGCGCCCTGGGCCCAGCTCTGTCCCACACCGCGGTCACATGGCACCACCTCTCTTGCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTGCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGGTGAGAGGCCAGCACAGGGAGGGAGGGTGTCTGCTGGAAGCCAGGCTCAGCGCTCCTGCCTGGACGCATCCCGGCTATGCAGCCCCAGTCCAGGGCAGCAAGGCAGGCCCCGTCTGCCTCTTCACCCGGAGGCCTCTGCCCGCCCCACTCATGCTCAGGGAGAGGGTCTTCTGGCTTTTTCCCCAGGCTCTGGGCAGGCACAGGCTAGGTGCCCCTAACCCAGGCCCTGCACACAAAGGGGCAGGTGCTGGGCTCAGACCTGCCAAGAGCCATATCCGGGAGGACCCTGCCCCTGACCTAAGCCCACCCCAAAGGCCAAACTCTCCACTCCCTCAGCTCGGACACCTTCTCTCCTCCCAGATTCCAGTAACTCCCAATCTTCTCTCTGCAGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGGTAAGCCAGCCCAGGCCTCGCCCTCCAGCTCAAGGCGGGACAGGTGCCCTAGAGTAGCCTGCATCCAGGGACAGGCCCCAGCCGGGTGCTGACACGTCCACCTCCATCTCTTCCTCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGTGGGACCCGTGGGGTGCGAGGGCCACATGGACAGAGGCCGGCTCGGCCCACCCTCTGCCCTGAGAGTGACCGCTGTACCAACCTCTGTCCCTACAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTGAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCCTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGCGACGGCCGGCAAGCCCCCGCTCCCCGGGCTCTCGCGGTCGCACGAGGATGCTTGGCACGTACCCCCTGTACATACTTCCCGGGCGCCCAGCATGGAAATAAAGCACCCAGCGCTGCCCTGGGCCCCTGCGAGACTGTGATGGTTCTTTCCACGGGTCAGGCCGAGTCTGAGGCCTGAGTGGCATGAGGGAGGCAGAGCGGGTCCCACTGTCCCCACACTGGCCCAGGCTGTGCAGGTGTGCCTGGGCCCCCTAGGGTGGGGCTCAGCCAGGGGCTGCCCTCGGCAGGGTGGGGGATTTGCCAGCGTGGCCCTCCCTCCAGCAGCACCTGCCCTGGGCTGGGCCACGGGAAGCCCTAGGAGCCCCTGGGGACAGACACACAGCCCCTGCCTCTGTAGGAGACTGTCCTGTTCTGTGAGCGCCCCTGTCCTCCCGACCTCCATGCCCACTCGGGGGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTACCGAGCTCGAATTCATCGATGATATGAGATCTGCCGGTCTCCCTATAGTGAGTCGTATTAATTTCGATAAGCCAGGTTAACCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGGACATATTGTCGTTAGAACGCGGCTACAATTAATACATAACCTTATGTATCATACACATACGATTTAGGTGACACTATA O-548 (SEQ ID NO: 221)ATGGTCCCAGCTCAGCTCCTCGGTCTCCTGCTGCTCTGGTTCCC O-549 (SEQ ID NO: 222)AGGTTCCAGATGCGACATCCAGATGACCCAGTCTCCATCTTCCG O-550 (SEQ ID NO: 223)TGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCG O-551 (SEQ ID NO: 224)AGTCAGGATATTAGCAGCTGGTTAGCCTGGTATCAGCATAAACC O-552 (SEQ ID NO: 225)AGGTAAAGCACCTAAGCTCCTGATCTATGCTGCATCCAGTTTGC O-563 (SEQ ID NO: 226)AGGAGCTTAGGTGCTTTACCTGGTTTATGCTGATACCAGGCTAA O-564 (SEQ ID NO: 227)CCAGCTGCTAATATCCTGACTCGCCCGACAAGTGATGGTGACTC O-565 (SEQ ID NO: 228)TGTCTCCTACAGATGCAGACACGGAAGATGGAGACTGGGTCATC O-566 (SEQ ID NO: 229)TGGATGTCGCATCTGGAACGTGGGAACCAGAGCAGCAGGAGACC O-567 (SEQ ID NO: 230)GAGGAGCTGAGCTGGGACCATCATGGTGGCAAGCTTAGAGTC O-527 (SEQ ID NO: 231)GACTCTAAGCTTGCCACCATGATGGTCC O-562 (SEQ ID NO: 232)ACCTTGATGGGACACCACTTTGCAAACTGGATGCAGCATAGATC O-553 (SEQ ID NO: 233)AAAGTGGTGTCCCATCAAGGTTCAGCGGAAGTGGATCTGGGACA O-554 (SEQ ID NO: 234)GATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGC O-555 (SEQ ID NO: 235)AACTTACTATTGTCAACAGGCTAATAGTTTCCCGTACACTTTTG O-556 (SEQ ID NO: 236)GTCAGGGAACCAAGCTGGAGATCAAACGAACTGTGGCTGCACCA O-557 (SEQ ID NO: 237)TCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGA O-558 (SEQ ID NO: 238)GGGAAGATGAAGACAGATGGTGCAGCCACAGTTCGTTTGA O-559 (SEQ ID NO: 239)TCTCCAGCTTGGTTCCCTGACCAAAAGTGTACGGGAAACTATTA O-560 (SEQ ID NO: 240)GCCTGTTGACAATAGTAAGTTGCAAAATCTTCAGGCTGCAGGCT O-561 (SEQ ID NO: 241)GCTGATGGTGAGAGTGAAATCTGTCCCAGATCCACTTCCGCTGA O-493 (SEQ ID NO: 242)TCAACTGCTCATCAGATGGC pLC6G5 (SEQ ID NO: 243)TCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCAflCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGCCAAGCTAGCGGCCGCGGTCCAACCACCAATCTCAAAGCTTGCCACCATGATGGTCCCAGCTCAGCTCCTCGGTCTCCTGCTGCTCTGGTTCCCAGGTTCCAGATGCGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGATATTAGCAGCTGGTTAGCCTGGTATCAGCATAAACCAGGTAAAGCACCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGTGTCCCATCAAGGTTCAGCGGAAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAATAGTTTCCCGTACACTTTTGGTCAGGGAACCAAGCTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGAGGGAGAAGTGCCCCCACCTGCTCCTCAGTTCCAGCCTGACCCCCTCCCATCCTTTGGCCTCTGACCCTTTTTCCACAGTGGACCTACCCCTATTGCGGTCCTCCAGCTCATCTTTCACCTCACCCCCCTCCTCCTCCTTGGCTTTAATTATGCTAATGTTGGAGGAGAATGAATAAATAAAGTGAATCTTTGCACCTGTGGTTTCTCTCTTTCCTCAATTTAATAATTATTATCTGTTGTTTACCAACTACTCAATTTCTCTTATAAGGGACTAAATATGTAGTCATCCTAAGGCGCATAACCATTTATAAAAATCATCCTTCATTCTATTTTACCCTATCATCCTCTGCAAGACAGTCCTCCCTCAAACCCACAAGCCTTCTGTCCTCACAGTCCCCTGGGCCATGGATCCTCACATCCCAATCCGCGGCCGCAATTCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCACTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGG CGGTTTGCGTATTGGGCGCO-528 (SEQ ID NO: 244) TTCTTCCTCCTCCTGGTGGCAGCTCCTAGATGGGTCCTGTCTC O-529(SEQ ID NO: 245) AGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTC O-530 (SEQ.ID NO: 246) GGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGTTCCTTC O-531 (SEQ IDNO: 247) AGTGGTTACTACTGGAGCTGGATCCGCCAGCCACCAGGTAAGG O-532 (SEQ ID NO:248) GTCTGGAGTGGATTGGTGAAATCAATCATAGTGGAAGCACCAA O-543 (SEQ ID NO: 249)TTCACCAATCCACTCCAGACCCTTACCTGGTGGCTGGCGGATC O-544 (SEQ ID NO: 250)CAGCTCCAGTAGTAACCACTGAAGGAACCACCATAGACAGCGC O-545 (SEQ ID NO: 251)AGGTGAGGGACAGGGTCTCCGAAGGCTTCAACAGTCCTGCGCC O-548 (SEQ ID NO: 252)CCACTGCTGTAGCTGCACCTGAGACAGGACCCATCTAGGAGCT O-547 (SEQ ID NO: 253)GCCACCAGGAGGAGGAAGAACCACAGGTGTTTCATGGTGGCAAGCTTG O-496 (SEQ ID NO: 254)CATGAAACACCTGTGGTTCTTCC O-542 (SEQ ID NO: 255)TCTTGAGAGACGGGTTGTAGTTGGTGCTTCCACTATGATTGAT O-533 (SEQ ID NO: 256)CTACAACCCGTCTCTCAAGAGTCGAGTCACCATATCAGTAGAC O-534 (SEQ ID NO: 257)ACGTCCAAGAACCAGTTCTCTCTGAAACTGAGCTCTGTGACCG O-535 (SEQ ID NO: 258)CTGCGGACACGGCTGTGTATTACTGTGCGAGAGTAATTAATTG O-536 (SEQ ID NO: 259)GTTCGACCCTTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA O-537 (SEQ ID NO: 260)GCCTCAACCAAGGGCCCATCGGTCTTCCCCCTGGCACC O-539 (SEQ ID NO: 262)CCTGGCCCCAAGGGTCGAACCAATTAATTACTCTCGCACAGTA O-540 (SEQ ID NO: 263)ATACACAGCCGTGTCCGCAGCGGTCACAGAGCTCAGTTTCAGA O-541 (SEQ ID NO: 264)GAGAACTGGTTCTTGGACGTGTCTACTGATATGGTGACTCGAC O-538 (SEQ ID NO: 261)CGATGGGCGCTTGGTTGAGGCTGAGGAGACGGTGACCAGGGTTC O-490 (SEQ ID NO: 265)GAAGCACCAACTACAACCCG O-520 (SEQ ID NO: 266) GAGTTCCACGACACCGTCACC O-521(SEQ ID NO: 267) GACCTCAAGCTTGCCACCATGAAACACCTGTGG pHC6G5 (SEQ ID NO:268) GAACTCGAGCAGCTGAAGCTTGCCACCATGAAACACCTGTGGTTCTTCCTCCTCCTGGTGGCAGCTCCTAGATGGGTCCTGTCTCAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGTTCCTTCAGTGGTTACTACTGGAGCTGGATCCGCCAGCCACCAGGTAAGGGTCTGGAGTGGATTGGTGAAATCAATCATAGTGGAAGCACCAACTACAACCCGTCTCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCTCTGAAACTGAGCTCTGTGACCGCTGCGGACACGGCTGTGTATTACTGTGCGAGAGTAATTAATTGGTTCGACCCTTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCAACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGGTGAGAGGCCAGCACAGGGAGGGAGGGTGTCTGCTGGAAGCCAGGCTCAGCGCTCCTGCCTGGACGCATCCCGGCTATGCAGCCCCAGTCCAGGGCAGCAAGGCAGGCCCCGTCTGCCTCTTCACCCGGAGGCCTCTGCCCGCCCCACTCATGCTCAGGGAGAGGGTCTTCTGGCTTTTTCCCCAGGCTCTGGGCAGGCACAGGCTAGGTGCCCCTAACCCAGGCCCTGCACACAAAGGGGCAGGTGCTGGGCTCAGACCTGCCAAGAGCCATATCCGGGAGGACCCTGCCCCTGACCTAAGCCCACCCCAAAGGCCAAACTCTCCACTCCCTCAGCTCGGACACCTTCTCTCCTCCCAGATTCCAGTAACTCCCAATCTTCTCTCTGCAGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGGTAAGCCAGCCCAGGCCTCGCCCTCCAGCTCAAGGCGGGACAGGTGCCCTAGAGTAGCCTGCATCCAGGGACAGGCCCCAGCCGGGTGCTGACACGTCCACCTCCATCTCTTCCTCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCAGCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGTGGGACCCGTGGGGTGCGAGGGCCACATGGACAGAGGCCGGCTCGGCCCACCCTCTGCCCTGAGAGTGACCGCTGTACCAACCTCTGTCCCTACAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGGCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGCGACGGCCGGCAAGCCCCCGCTCCCCGGGCTCTCGCGGTCGCACGAGGATGCTTGGCACGTACCCCCTGTACATACTTCCCGGGCGCCCAGCATGGAAATAAAGCACCCAGCGCTGCCCTGGGCCCCTGCGAGACTGTGATGGTTCTTTCCACGGGTCAGGCCGAGTCTGAGGCCTGAGTGGCATGAGGGAGGCAGAGCGGGTCCCACTGTCCCCACACTGGCCCAGGCTGTGCAGGTGTGCCTGGGCCCCCTAGGGTGGGGCTCAGCCAGGGGCTGCCCTCGGCAGGGTGGGGGATTTGCCAGCGTGGCCCTCCCTCCAGCAGCACCTGCCCTGGGCTGGGCCACGGGAAGCCCTAGGAGCCCCTGGGGACAGACACACAGCCCCTGCCTCTGTAGGAGACTGTCCTGTTCTGTGAGCGCCCCTGTCCTCCCGACCTCCATGCCCACTCGGGGGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTACCGAGCTCGAATTCATCGATGATATCAGATCTGCCGGTCTCCCTATAGTGAGTCGTATTAATTTCGATAAGCCAGGTTAACCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGGCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGrAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTGCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTGTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCGCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAAGTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGGACATATTGTCGTTAGAACGCGGCTACAATTAATACATAACCTTATGTATCATACACATACGATTTAGGTGACACTATA HC6G5 (SEQ ID NO: 219)AAGCTTGCCACCATGAAACACCTGTGGTTCTTCCTCCTCCTGGTGGCAGCTCCTAGATGGGTCCTGTCTCAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGTTCCTTCAGTGGTTACTACTGGAGCTGGATCCGCCAGCCACCAGGTAAGGGTCTGGAGTGGATTGGTGAAATCAATCATAGTGGAAGCACCAACTACAACCCGTCTCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCTCTGAAACTGAGCTCTGTGACCGCTGCGGACACGGCTGTGTATTACTGTGCGAGAGTAATTAATTGGTTCGACCCTTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCAACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGGACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGT LC6G5 (SEQ ID NO: 220)AAGCTTGCCACCATGATGGTCCCAGCTCAGCTCCTCGGTCTCCTGCTGCTCTGGTTCCCAGGTTCCAGATGCGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGATATTAGCAGCTGGTTAGCCTGGTATCAGCATAAACCAGGTAAAGCACCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGTGTCCCATCAAGGTTCAGCGGAAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAATAGTTTCCCGTACACTTTTGGTCAGGGAACCAAGCTGGAGATCAAACGAACTG TGGCTGCACCATCTGTCTTC

Example 43 Binding of Human Anti-CD4 Monoclonal Antibodies to Non-HumanPrimate Lymphocytes

It is desirable to be able perform preclinical toxicology andpharmacokinetic studies of human anti-CD4 monoclonal antibodies inanimal models. It is further desirable for some purposes that the animalbe a non-human primate that expresses CD4 comprising a cross-reactiveepitope with human CD4 such that it is recognized by the monoclonalantibody. Three different non-human primate species, chimpanzee, rhesus,and cynomolgus monkeys, were tested for cross-reactive CD4 epitopes withthe 5 different human anti-CD4 monoclonal antibodies from hybridomas1E11, 1 G2, 6G5, 10C5, and 4D1. Peripheral blood lymphocytes wereisolated from whole blood of chimpanzee, rhesus, and cynomolgus monkeys.The isolated cells were double stained with human antibody from each ofthese 5 hybridomas (detected with FITC-anti-human IgG) and PE-anti-CD8or PE-anti-CD4. The stained cells were then analyzed by flow cytometryto determine if each of the human monoclonal antibodies bound toendogenous CD4 on the surface of lymphocytes from each of these threenon-human primates. Four of the five antibodies, 1E11, 6G5, 10C5, and4D1, were found to bind to chimpanzee CD4 cells. Additionally, four ofthe five antibodies, 6G5, 1G2, 10C5, and 4D1, were found to bind to bothrhesus and cynomolgus CD4 cells. Thus, three of five antibodies, 6G5,10C5, and 4D1, bind to CD4 cells in each of the three non-human primatespecies tested.

Example 44 Lack of Correlation Between Modulation and Non-Depletion

There are no known in vitro assays that can reliably predict whether amonoclonal antibody (mAb) will be nondepleting or immunosuppressive inpatients. However, a correlation has been observed between the abilityof three different mAbs to deplete (or not deplete) in humans andnonhuman primates such as chimpanzees and cynomolgus monkeys (See, e.g.,M. Jonker et al., Clin. Exp. Immunol., 93:301-307 (1993); and J. A.Powelson et al., Transplantation, 57:788-793 (1994)). Therefore a studywas performed using human mAbs in nonhuman primates.

Chimpanzees were used in this study, because one of the anti-CD4 mAbs,1E11, recognizes CD-4 only in chimpanzees and not in Rhesus orcynomolgus monkeys. A second mAb, 6G5, recognizes CD4 in chimpanzees,Rhesus and cynomolgus monkeys. A third mAb, 1G2, does not recognize CD4in chimpanzees, but does in Rhesus and cynomolgus monkeys. That mab hasalready been shown to be nondepleting in vivo in cynomolgus monkeys.

In addition to examining the effect of human mAbs on CD4+ T cell numbersin peripheral blood, the effect of the mAb administration on in vivo Tcell function was also evaluated. The most accepted manner to do this isto use animals that have been presensitized to an antigen such astuberculin or tetanus toxoid and who will mount a hypersensitivityreaction in the skin.

Three male chimpanzees were enrolled in this study. Baseline whole bloodsamples were obtained on days −7, −3 and 1. After the blood draw on day1, one chimpanzee each was intravenously infused with one of the twohuman mAbs (1E11 or 6G5) at 2 mg/kg. The third chimpanzee received anequal volume/kg of buffer only. Blood was drawn at 30 mins, 2 hrs, 8hrs, 24 hrs and 48 hrs post-infusion. On day 2, a skin reactivity testwas performed.

Results shown in Table 25 below clearly demonstrate that 1E11 causedtransient depletion of peripheral lymphocytes, with most CD4+ T cellsbeing depleted. Even though 6G5 did not cause lymphocyte or CD4+ T celldepletion, both mAbs were able to inhibit a hypersensitivity response totetanus toxoid, compared to the control chimpanzee. Thus, both humanmAbs appear to be immunosuppressive in vivo, and this immunosuppressiondoes not necessarily require T cell depletion.

TABLE 25 Effect of Human mAbs on Peripheral Chimpanzee LymphocytesPeripheral Lymphocytes (million/ml) Study Day 1E11 6G5 Control −7 4.26.4 4.2 −4 4.0 9.9 4.4 1, pre-infusion 4.8 5.7 5.8 1, 30 min post 1.66.0 4.0 1, 2 hr post 1.0 6.7 5.2 1, 6 hr post 1.5 8.0 4.2  2 3.5 9.6 5.7 3 3.9 9.7 5.9Whole blood samples from the chimpanzees were collected on study days 5,8, 15 and 29. PBMC from all blood samples were isolated and examined byflow cytometry to determine the percent of CD4 T, CD45RA/CD4 T (naive),CD45RO/CD4 T (memory), CD8 T and CD19 B cells present in PBMC; thedensity of CD4 molecules per cell; and whether the human mAbs had boundto those cells. The cells were prepared for flow cytometry as described(Fishwild et al., 1996, Nature Biotechnology 14:845-851). Complete bloodcounts were performed on a sample of whole blood to quantitatelymphocytes. The total number of cells for any given subset was thenderived by multiplying the percent of such cells by the total number oflymphocytes.

The CD4⁺ cells were determined from positive staining of PBMC withPE-OKT4 (Fisher) and from negative staining with FITC-CD8 (CalTag). The% CD3⁺, CD8⁻, CD45RA⁺ (naive CD4⁺ T) cells and the % CD3⁺, CD8⁻, CD45RO⁺(memory CD4⁺ T) cells were determined from PBMC stained withTriColor-CD8 (CalTag), FITC-Leu4a (Becton-Dickinson) and PE-CD45RA(CalTag) or with TriColor-CD8, FITC-Leu4a and PE-CD45RO (CalTag),respectively. The total number of CD4⁺ cells (top left) and the ratio ofnaive to memory CD4 cells (top right) were then calculated. The amountof CD4 (bottom left) was determined from the mean channel fluorescenceof PE-OKT4⁺ lymphocytes. The amount of human mAb bound to CD4⁺ (PE-BF5⁺,SeroTec) cells (bottom right) was determined from the mean channelfluorescence (MCF) of FITC-goat anti-human Fcγ+ cells. Results obtainedfor gated lymphocytes only are shown here.

As shown in FIG. 91, only 1E11 and not 6G5 depleted CD4⁺ T cells incirculation, even though both bound equally well to CD4⁺ T cells.(Depletion refers to loss or decrease in numbers of specific immunecells from circulation, from lymphoid tissues, or from both.) Asexpected, 1E11 strongly and 6G5 weakly modulated CD4 antigen on T cellsin vivo (FIG. 91). The slightly lower binding of 1E11 to CD4⁺ cells ascompared to 6G5 seen from 30 min through 2 days is probably aconsequence of the greater CD4 modulation observed with 1E11.(Modulation refers to loss of antigen from the surface of a cell, or adecrease in surface antigen density. The loss can occur as a result ofshedding or internalization of the antigen, usually in the form ofantigen-antibody complexes.) Memory and naive CD4⁺ cells were equallyaffected by 1E11. Not unexpectedly, 1E11 depleted monocytes (which areCD4⁺) until day 5, whereas 6G5 did not.

There was transient and nonspecific depletion of CD8⁺ cells only in the1E11 animal (FIG. 92), whereas CD19⁺ B cells were not affected in any ofthe chimpanzees. The total number of CD8⁺, CD3⁺ (T suppressor) cells(left) and the total number of CD19⁺ cells (right) were determined fromPBMC co-stained with PE-CD8 and FITC-Leu4a or stained with FITC-CD19(CalTag), respectively.

In summary, 1E11 which induced extensive CD4 modulation both in vitroand in vivo, unexpectedly depleted CD4⁺ T cells. 6G5 which inducesmoderate CD4 modulation both in vitro and in vivo, unexpectedly did notdeplete CD4⁺ T cells. Thus, the seeming correlation between modulationand nondepletion that had been observed for other anti-CD4 mAbs has beendisproven.

Example 45 Prevention of T-Helper Dependent Immune Response In Vivo

The animals described in Example 44 were immunized by injection of 1.5ml of tetanus toxoid (TT, Fort Dodge Animal Health Care, Fort Dodge,Iowa) intramuscularly in the thigh on day −21. The animals received 0.1ml of a 10% TT solution or saline alone intradermally on the back ondays 2 and 29. The site was examined 24 hrs later. Reactions were scoredas 0 if there was no change at the site of injection, 1 if the injectionsite was reddened, 2 if the injection site was reddened and raised, and3 if the injection site was reddened, raised and firm. On day 2, inchimpanzees receiving either of the two human mAbs, 1E11 or 6G5, therewas no injection site reaction to TT while the chimpanzee receiving thePBS injection had a reddened injection site. On day 29, all chimpanzeeshad reddened injection site reactions for TT. There was no reaction atthe site where saline had been injected on either day. Thus, while thehuman mAbs were present (on day 2), they apparently were able to preventa T-helper dependent immune response in vivo.

Example 46 Depletion Studies in the Cynomolaus Monkey

Depletion studies were also carried out in the cynomolgus monkey. Abaseline whole blood sample was obtained on day 0 from four cynomolgusmonkeys. After this blood draw, one cynomolgus monkey each wasintravenously infused with one of the three mabs (1E11, 6G5 or 1G2) at 2mg/kg. The fourth cynomolgus monkey received the same volume/kg of PBSonly. Blood was drawn at 2 hrs and 8 hrs post-infusion on day 0. Bloodwas drawn daily (approx. every 24 hrs after the infusion) on days 1, 2,4, 7, 11, 18 and 32. PBMC from whole blood were isolated over Ficollfollowing standard procedures. Aliquots of the PBMC were then stained asin Example 44 with various fluorochrome monoclonal antibody conjugatesto monitor CD4⁺ T cells and CD8⁺ T cells, and human monoclonal antibodybound to CD4⁺ T cells.

As described in Example 43, in this species, 1E11 mAb does not recognizeCD4, whereas both 6G5 and 1G2 mAbs do. Thus, 1E11 serves as a negativecontrol human mAb in the cynomolgus study.

As shown in FIG. 93, none of the antibodies depleted CD4⁺ T cellsincluding the poorly modulating 6G5 mAb. Nor was there any effect onCD8⁺ T cells. Both 6G5 and 1G2 bound to CD4⁺ T cells, but only 1G2induced CD4 modulation in vivo. Moreover, although both 1G2 and 1E11extensively modulated CD4 in vivo, one mAb was depleting and one wasnot. Therefore, there appears to be no correlation between modulationand depletion. In FIG. 93, the percent of CD4⁺ cells (top left) and thepercent of CD8⁺ cells (top right) are shown. The CD4⁺ cells weredetermined from positive staining of PBMC with PE-OKT4 (Ortho) and fromnegative staining with FITC-CD8 (Becton-Dickinson). The amount of CD4(bottom left) was determined from the MCF of PE-OKT4⁺ lymphocytes. Theamount of human mAb bound to CD4⁺ (PE-OKT4⁺) cells (bottom right) wasdetermined from the MCF of FITC-goat anti-human Fcγ⁺ cells. Resultobtained for gated lymphocytes only are shown.

Example 47 Lymph Node Lymphocytes

To determine whether the human mAbs were able to exit the vasculatureand appear in other peripheral organs, as part of the previouslydescribed chimpanzee study, inguinal lymph nodes were examined on days−7, 2 and 29. Biopsies were performed and single cell suspensions inmedium were prepared. The percent of CD4, CD8 and CD3 cells present inthe lymph node mononuclear cells was determined, and, using the methodsdescribed in Examples 43-45, it was determined whether the human mAbshad bound to the lymph node mononuclear cells.

As shown in Table 26 and FIG. 94, both 1E11 and 6G5 slightly depletedCD4⁺ T cells in lymph nodes on day 2. This effect was reversed on day 29for 6G5 and became even more apparent for 1E11. There was acorresponding increase in CD8⁺ T cells on all days when CD4⁺ T cellswere depleted. Part, but not all, of the explanation for decreased CD4⁺T cells on day 29 in chimpanzee A008 may be due to a lowered percent ofCD3⁺ cells (and concomitantly increased percent of B cells). Both 1E11and 6G5 bound to CD4⁺ T cells in lymph nodes. Thus, these two mAbs wereable to exit the blood and enter lymph tissue. Whether they did so assoluble antibodies or attached to CD4 cells can not be determined fromthese data. 1E11 modulated CD4 antigen in lymph nodes, whereas 6G5minimally modulated CD4, if at all. This effect was observed only on day2.

In FIG. 94, the % CD4⁺ T cells (top left) and on the % CD8⁺, CD3⁺ Tcells (top right) were determined from positive staining of lymph nodelymphocytes with PE-OKT4 and from negative staining with FITC-CD8 orfrom lymphocytes co-stained with PE-CD8 and FITC-Leu4a. The amount ofCD4 (bottom left) as determined from the MCF of PE-OKT4⁺ lymphocytes isshown. The amount of human monoclonal antibody bound to CD4⁺ (PE-BF5⁺)cells (bottom right) was determined from the MCF of FITC-goat anti-humanFcγ⁺ cells.

TABLE 26 Summary of Flow Cytometry Studies on Lymph Node LymphocytesChimpanzee Number (Article Injected) Cell Type A008 (1E11) A010 (6G5)A017 (PBS) % CD4⁺ Decr on d2, d29 Decr on d2 % CD8⁺ Incr on d2, d29 Incron d2 % CD3⁺ Decr on d29 % CD19⁺ Incr on d29 MCF CD4 Decr on d2 MCFHuMAb Incr on d2 Incr on d2 Blanks mean that there was no significantchange in the parameter being examined. Decr = decrease Incr = increase

In summary, it is apparent from these data that human mabs could migrateinto lymphoid tissue. These mabs were cleared from the lymph nodesbetween days 2 and 29. When present in the lymph node, 1E11 induced CD4modulation, although to a lesser extent than in peripheral blood on thesame study day. One of these antibodies, 1E11, depleted CD4⁺ T cellsfrom lymph nodes on day 2 and 29. Thus, 1E11 appears to be able todeplete CD4⁺ T cells from peripheral organs for a longer period of timethan it does from peripheral blood.

Example 48 Half Life of Human Monoclonal Antibodies in Nonhuman Primates

As part of the cynomolgus monkey study (described in Example 46), plasmasamples were obtained at various time points and assayed for thepresence of human mAb. The standard quantitative rCD4 ELISA (Lonberg etal., 1994, Nature 368:856-9) was used, except that the cynomolgus plasmawas diluted 1:1000 and 1:10000 prior to assay and the mAb standards werediluted in 1: 1000 and 1:10000 normal cynomolgus plasma in diluentbuffer instead of the diluent buffer alone.

As shown in FIG. 95, the serum half-life of 1E11, the human mAb whichdoes not recognize cynomolgus CD4, was 10 days. The serum half-life ofthe human mAbs which do recognize cynomolgus CD4, 6G5 and 1G2, were 39and 14 hrs, respectively. Thus, it would appear that the presence of anantigen sink can significantly decrease the half-life of human mAbs andthat antigen turnover (as occurs with CD4 modulation) can furtherdecrease serum-half life. Thus, the most desirable mAb for chronicclinical use would be a non-depleting, non-modulating mAb such as 6G5.

Example 49 Effect on Response to Tetanus Toxoid

It has been shown above that all human IgG_(κ) anti-CD4 mAbs thatrecognized the membrane distal domains of CD4 were able to inhibitresponses to alloantigen (cell surface expressed MHC class II molecules)in vitro, as in a mixed lymphocyte reaction (MLR). The ability of thesehuman anti-CD4 mAbs to inhibit human cell responses to a soluble foreignantigen, tetanus toxoid (TT), was then determined in vitro. PBMC fromhuman donors were prescreened for reactivity to TT, and the reactivePBMC stored at −80° C. and subsequently assayed for antibody inhibitionusing freshly thawed PBMC from those responsive donors. Serial dilutionsof human monoclonal antibodies were added to 1×10⁵ PBMC per well inmedium followed by 5 LF/ml of TT (Wyeth-Ayerst). Cells were cultured fora total of 7 days and ³H-thymidine was added 16 hr prior to harvest.

As shown in FIG. 96, all three anti-CD4 mAbs tested inhibited responsesto TT. 1E11 antibody was much more potent than either 6G5 or 1G2. Thenegative control human mAb, 4E4, had no effect.

Example 50 Production of Human Anti-IL8 mAbs

Recombinant human IL8 (rhIL8) was expressed in E. coli using the pETsystem (Novagen) and purified to N-terminal sequence homogeneity usingheparin affinity chromotography (hi-trap, Pharmacia) followed by sizeexclusion chromotography (Superdex 75, Pharmacia) and C18 reverse phaseHPLC (ultrasphere ODS, Beckman). HuMAb transgenic mice were immunizedwith 20 ug recombinant human IL8 in complete Fruend's adjuvant, thenwith 4-5 weekly injections of 5 ug rhIL8 in incomplete Fruend'sadjuvant. In the first and second round of immunizations, transgenicmice from three different strains were used (five HCo7/KCo4 mice, nineHC2/KCo5 mice, and three HC2/HCo7/KCo4/KCo5 mice; all were JhD/JkD). Inthe third round of immunizations, transgenic mice from two differentstrains were used (four HC2/KCo5 mice, and six HCo7/KCo5 mice; all wereCmD/JkD). Serum was removed prior to each immunization and tested forthe presence of human IgG and IgM antibodies by ELISA. Briefly, wells ofmicrotiter plates were coated with 0.2 ug/ml rhIL8 in carbonate bufferovernight at 4° C., blocked with 5% chick serum in PBS for 2 hrs at 37°C., incubated with goat anti-human IgG or IgM for 1 hr at 37° C., andfinally incubated with ABTS substrate for 1 hr at 25° C. Plates werewashed extensively between each step.

All transgenic mice from all strains responded readily to immunizationwith rhIL8, with human IgM antibodies detected prior to human IgGantibodies in serum. Human IgG responses peaked around week 4 and didnot increase with further immunizations as shown in FIG. 97. In general,mice containing the HCo7 transgene responded slightly better than thosecontaining only the HC2 transgene. This may be related to the particularV regions present in these two transgenes (of the four Vh regions, onlyone is common to both transgenes) or to the site of intergration or tosome other as yet undetermined factor. In any case, the difference inresponsiveness was small.

Spleens from four mice (two were from HC2/HCo7/KCo4/KCo5/JhD/JkD miceand two were from HCo7/KCo5/CmD/JkD mice) were removed and processed forfusion by as described (Fishwild et al. 1996, Nature Biotechnology14:845-851). These mice were chosen because they had the highest titersof human IgG anti-IL8 antibodies in serum. A total of 29 hybridomassecreting human IgGk anti-rhIL8 mAbs were detected in parental wells. Ofthe 21 parental hybridomas attempted, 18 of these hybridomas weresuccessfully subcloned. There was no significant difference betweenthese two transgenic strains with respect to either the total number ofparental hybridomas detected or to their subcloning efficiency.

Example 51 Characterization of Human IgG_(κ) Anti-IL8 mAbs

The first 14 subcloned hybridomas obtained and their secreted mAbs wereextensively characterized (all obtained from HC2/HCo7/KCo4/KCo5/JhD/JKDmice). The other 4 subcloned hybridomas have been partiallycharacterized.

The amount of mAb secreted by each hybridoma was determined by aquantitative ELISA as previously described (Lonberg et al., 1994, Nature368:856-9). The on- and off-rates for human mAbs were also determined asdescribed (Lonberg et al., 1994, Nature 368:856-9), except that rhIL8was coupled to the BIAcore sensor chip instead of rCD4.

As shown in Table 27, most of these hybridomas secreted very highamounts of human antibody (>10 μg/ml), ranging from 0.5 to 32 μg/ml.Most of these mAbs had very high binding avidities, with Ka values >10⁹M⁻¹. There was quite a large range of on-rates (k_(assoc)) and off-rates(k_(dissoc)). Moreover, many of the on- and off-rates are unique,suggesting that among these 14 mAbs, there may be at least 10 distinctmAbs (i.e., not derived from the same parental splenic B cell).

TABLE 27 mAb Secretion, Avidity and Rate Constants Human mAb mAb ConcRate Constants (mouse #)  (mg/ml) k_(assoc) (M⁻¹s⁻¹) k_(dissoc) (s⁻¹) Ka(M⁻¹) 1F8.19 17 2.7 × 10⁴ 1.5 × 10⁻⁵ 1.8 × 10⁹ (14476) 2D11.8 1.8 4.6 ×10⁵ 8.1 × 10⁻⁵ 5.6 × 10⁹ 2D11.6 1.2 5.3 × 10⁵ 2.2 × 10⁻⁵ 2.3 × 10¹⁰2F9.5 1.2 4.4 × 10⁵ 3.0 × 10⁻⁵ 1.5 × 10¹⁰ 2F9.4 1.0 4.4 × 10⁵ 1.4 × 10⁻⁵3.2 × 10¹⁰ 2G1.8 1.3 4.9 × 10⁵ 5.7 × 10⁻⁵ 8.7 × 10⁹ 3E5.4 26 1.3 × 10⁵4.3 × 10⁻⁵ 3.1 × 10⁹ 5E7.4 0.5 nd nd 5F10.6 11 4.6 × 10⁴ 5.2 × 10⁻⁶ 8.8× 10⁹ 5H8.8 16 7.7 × 10⁴ 6.3 × 10⁻⁵ 1.2 × 10⁹ 2C6.1 32 9.9 × 10⁴ 2.7 ×10⁻⁵ 3.6 × 10⁹ (14477) 2D6.3 6.3 9.7 × 10⁴ 4.6 × 10⁻⁵ 2.1 × 10⁹ 3A1.7 201.2 × 10⁵ 1.5 × 10⁻⁴ 8.2 × 10⁸ 4D4.8 14 6.3 × 10⁴ 6.7 × 10⁻⁵ 9.4 × 10⁸7C5.7 20 4.0 × 10⁴ 1.5 × 10⁻⁴ 2.6 × 10⁸ 10A6.9 6.2 1.0 × 10⁵ 7.0 × 10⁻⁵1.5 × 10⁹ The rate and equilibrium constants for monoclonal antibodies(mAb) were determined with a BIAcore, using antigen (rhIL8) coupled tothe sensor chip and flowing mAb over. These constants were derived fromone experiment using mAbs in spent tissue culture medium. nd = notdetermined

The mAbs were then tested for specificity. Various CXC chemokines (IL8,IP10, Nap2, ENA78, GROα, GRO.beta. and GROγ) were absorbed to ELISAplates and the binding of the human mAbs to those chemokines detected byan enzyme conjugated anti-human Ig antiserum as described in Example 49for the IL8 ELISA. Specificity as well as the ability to neutralize IL8was determined by examining whether the human mAbs could inhibit 1) thebinding of radiolabeled IL8 to IL8RA-expressing transfectants, 2) thebinding of radiolabeled IL8 to neutrophils, 3) the binding ofradiolabeled GROα to IL8RB-expressing transfectants and 4) the effect onIL8-induced Ca⁺⁺ flux in neutrophils.

Stable IL8RA-expressing transfectants were created using the murinepre-B lymphoma cell line (L1-2) as described (Campbell et al., 1996, J.Cell Biol. 134:255-66; Ponath et al., 1996, J. Exp. Med. 183:2437-48).All expression constructs were made in pcDNA3 (Invitrogen, CA).Wild-type IL-8RA and IL-8RB cDNA were subcloned into Hind III-Not I andEcoR I-Not I sites, respectively. The second initiation site in theIL-8RB sequence (which corresponds to amino acid sequences of MESDS (SEQID NO: 417) was used. Forty-eight hours post-transfection, 0.8 my/mlG418 was added and serial dilutions of cells plated in 96-well plates.After 1-2 weeks, G418-resistant cells were stained by appropriateanti-IL-8R antibodies. High level of expression was enriched either bylimiting dilution and re-screening or FACS sorting.

Ligand binding assays were carried out as follows. ¹²⁵I-labeled humanIL8 was purchased from Amersham (Arlington Heights, Ill.) or DuPont NEN(Boston, Mass.). For each binding reaction, 60-μl cell cells, washed andresuspended in HBSS containing 5% BSA at 5×10⁶/ml, was mixed with 60-μlof 2× reaction mix and incubated at 37° C. for 30 min. For most of theexperiments, the final concentration of ¹²⁵I-IL8 in the reaction was 0.1nM. Non-specific binding was determined in the presence of 100 nMunlabeled IL8. The binding reaction was stopped by transferring 100-μlof the mix to tubes containing 200-μl of Dibutyl phthalate/Bis2-ethylhexyl phthalate (1:1 mix) and spun at 12 K rpm for 3 min. Thesupernatant was frozen in dry ice and the cell pellet in the bottom ofthe tubes was cut off and subjected to counting in a gamma-counter. ForScatchard analysis, 0.05 nM of ¹²⁵I-IL8 was added with increasingconcentrations of unlabeled IL8. The data were curve fitted and K_(d)and B_(max) was calculated using the computer program Ligand (Munson etal., 1980, Anal. Biochem. 107, 220-239).

For the Ca⁺⁺ flux assay, neutrophils at 10⁷ cells/ml were incubated withthe fluorochrome Fluo-3 (Molecular Probes) for 30 at RT, washed twiceand resuspended at 2×10⁶ cells/ml. IL8 with or without mAbs was added toFluo-3 labeled cells and the internal Ca⁺⁺ concentrations weredetermined by analyzing FL1 on a FACScan over time.

Of the 14 human anti-rhIL8 monoclonal antibodies isolated from 2 mice,10 were specific for rhIL8 and 3 crossreacted with other chemokines (1has not yet been tested). Nine of the 10 IL8-specific mAbs wereneutralizing (see Table 28). One of the mAbs, 7C5, that crossreactedwith other CXC chemokines was able to inhibit IL8 binding, but two othermAbs, 1F8 and 5F10, that only crossreacted with GROa did not inhibit IL8binding. mAbs which could inhibit IL8 binding to IL8RA transfectantscould also inhibit IL8 binding to neutrophils and could interfere withIL8-induced Ca⁺⁺ flux. None of the three mAbs which bound to GROα wereable to inhibit GROα binding.

Based on these specificity data, it appears that there are at leastthree different epitopes on IL8 being recognized. The primary one isunique to IL8, a second is shared between IL8 and GROα, and a third isfound more broadly distributed on CXC chemokines.

TABLE 28 Specificity and Characterization of Human Anti-IL8 mAbsSpecific Inhibits Inhibits Inhibits for Cross-Reacts rIL8RA PMN Ca⁺⁺ mAbIL8?* with? Binding? Binding? flux? 1F8 NO GROα NO NO NO (14476) 2D11YES YES YES NO 2F9 YES YES nd nd 2G1 YES YES nd nd 3E5 nd nd nd nd nd5E7 YES YES nd nd 5F10 NO GROα NO NO NO 5H8 YES YES nd nd 2C6 YES YESYES YES (14477) 2D6 YES nd nd nd 3A1 YES YES nd YES 4D4 YES YES nd nd7C5 NO IP10, YES nd nd GROα 10A6 YES YES nd nd 2A2** YES YES YES YES nomAb NO NO NO NO *HuMAbs were tested by ELISA for reactivity with IL8,GROα, GROβ, GROγ, IP10, ENA78 and NAP2 nd = not done **2A2 is a murineanti-hIL8 IgG monoclonal antibody that was used as a positive controlfor these assays. The negative control was no mAb.

The ability of one the human mAbs, 2C6, to inhibit IL8 was examined inmore detail. The mAb was purified from spent tissue culture fluid over aprotein A column and tested for its ability to inhibit neutrophilchemotaxis and neutrophil elastase release.

In the chemotaxis assay, HUVEC or ECV304 cells were adhered to asemi-permeable membrane. Human leukocytes were added to the chamber ontop of the filter and IL8 (with or without human mAb) was added to thebottom chamber. Cells which migrated through the endothelial cellsadhered to the filter into the bottom chamber were enumerated with aflow cytometer using forward and side scatters. For the elastase releaseassay, neutrophils were incubated with cytochalasin B followed by mAb orbuffer followed by the elastase substrate. Fluorescence was thenmeasured immediately and 10 min after the addition of IL8.

Human mAb 2C6 demonstrated dose-dependent inhibition of both IL8-inducedneutrophil chemotaxis and elastase release (see FIG. 98). The IC₅₀values for 2C6 were 270 ng/ml and 330 ng/ml, respectively. Bothchemotaxis and elastase release were completely inhibited by as littleas 1 μg/ml of mAb. This human mAb is slightly more potent than the mostpotent murine anti-IL8 mAb examined to date.

The foregoing description of the preferred embodiments of the presentinvention has been presented for purposes of illustration anddescription. They are not intended to be exhaustive or to limit theinvention to the precise form disclosed, and many modifications andvariations are possible in light of the above teaching. It will beapparent that certain changes and modifications may be practiced withinthe scope of the claims. All publications and patent applications hereinare incorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference. Commonly assignedapplications U.S. Ser. No. 08/728,463 filed Oct. 10, 1996, U.S. Ser. No.08/544,404 filed 10 Oct. 1995, U.S. Ser. No. 08/352,322, filed 7 Dec.1994, U.S. Ser. No. 08/209,741 filed 9 Mar. 1994, U.S. Ser. No.08/165,699 filed 10 Dec. 1993 and U.S. Ser. No. 08/161,739 filed 3 Dec.1993, which is a continuation-in-part of Ser. No. 08/155,301 filed 18Nov. 1993, WO92/03918, U.S. Ser. No. 07/810,279 filed 17 Dec. 1991, U.S.Ser. No. 07/853,408 filed 18 Mar. 1992, U.S. Ser. No. 07/904,068 filed23 Jun. 1992, U.S. Ser. No. 07/990,860 filed 16 Dec. 1992, WO93/12227,and U.S. Ser. No. 08/053,131 filed 26 Apr. 1993 are each incorporatedherein by reference.

1. A method for identifying a murine IgG isotype immunoglobulin moleculecomprising a human variable region that specifically binds to apredetermined antigen, wherein the immunoglobulin molecule is expressedby a B lymphocyte hybridoma clone, wherein the B lymphocyte hybridomaclone is derived from a fusion of: (i) a B lymphocyte from a transgenicmouse, wherein the genome of said transgenic mouse comprises inactivatedmurine variable regions and an unrearranged human heavy chainimmunoglobulin variable region operably linked to a mu constant regiongene segment, wherein the unrearranged heavy chain variable regioncomprises multiple human VH gene segments, multiple human D genesegments, and multiple human JH gene segments, and wherein the muconstant region is selected from the group consisting of: (a) a humanS-mu sequence and a human or mouse mu coding sequence; and (b) a mouseS-mu sequence and a human or mouse mu coding sequence; and (ii) amyeloma cell; and which method comprises: (i) incubating a firstantibody mixture derived from the B lymphocyte hybridoma clonecontaining the murine IgG isotype immunoglobulin molecule expressed bysaid B lymphocyte hybridoma clone with said antigen under conditionssufficient to allow for specific binding, (ii) modifying the incubationconditions to remove non-specifically bound antibodies, (iii) incubatinga second antibody mixture containing an anti-murine IgG antibodycomprising a marker under conditions sufficient to allow for specificbinding of the anti-murine IgG antibody to murine IgG antibody, (iv)modifying the incubation conditions to remove non-specifically boundanti-murine IgG antibodies, and (v) identifying the murine IgGimmunoglobulin molecule comprising a human variable region whichspecifically binds to the predetermined antigen by identifying thepresence of the marker.
 2. The method of claim 1, wherein saidanti-murine IgG antibody comprising a marker is a polyclonal antibody ora monoclonal antibody.
 3. The method of claim 1, wherein saidanti-murine IgG antibody comprising a marker is a goat anti-murine IgGantibody comprising a marker or a rabbit anti-murine antibody comprisinga marker.
 4. The method of claim 1, wherein said marker is selected fromthe group consisting of: enzymatic markers; fluorescent small moleculemarkers; and fluorescent protein markers.
 5. The method of claim 1,wherein said marker is selected from the group consisting of:horseradish peroxidase; FITC; and phycoerythrin.